Regulation of the immune response by subclasses of T lymphocytes. II. The effect of adult thymectomy upon humoral and cellular responses in mice

Eur. J. ImmunoL 1975.5: 337-343 Subclasses of T lymphocytes 337

Elizabeth Simpson" and H. Cantor*+

Clinical Research Centre, Harrow' and

Regulation of the immune response by subclasses of T lymphocytes

Department of Medicine. Haward wi=l 11. The effect of adult thymectomy upon humoral and cellular responses in mice School, and Children's Cancer Research

Center, Boston+ The immunologic consequences resulting from thymectomy in adult life were investigated. Primary humoral responses were not diminished shortly after adult thymectomy, as judged by responses of intact thymectomized mice as well as by the ability of spleen cells from such mice to transfer primary responses. How- ever, secondary humoral responses were substantially reduced in irradiated recipients of spleen cells from primed adult-thymectomized mice, suggesting that the thymus is required in adult life to maintain a population of cells important in the generation of immunologic helper memory. By contrast, small doses of anti- thymocyte serum, which primarily affect recirculating T cells, abrogated the primary humoral response, but allowed the subsequent development of immune memory.

Using a technique permitting in vitro sensitization of purified T cells to alloantigens, it was shown that adult thymectomy increases the ability of T cells to generate primary cytotoxic responses, but had little effect upon the development of cytotoxic T memory activity. These experiments suggest that in adult life the thymus maintains a regulatory population of T cells in peripheral tissues which suppress early T cell differentiation to cytotoxic effector cells and potentiates the development of immune memory.

1. Introduction 2. Materials and methods

Peripheral lymphoid tissues contain thymus derived or "T" lymphocytes which migrate from the thymus mainly during the neonatal period, and are maintained throughout the life of the animal [ 1-31. Neonatal thymectomy results in severe depletion of all peripheral T lymphocytes, and major defects in humoral and cellular immune responses [3,4]. By contrast, thymectomy in adult life has been reported to result in little detectable effect on levels of recirculating lymphocytes, suggesting that this population of T lymphocytes is long lived. It has also been suggested that adult thymectomy has little effect upon immune function since the primary immune response is unimpaired up to a year after surgery [ 5-71.

There is, however, increasing evidence that a portion of T lymphocytes with distinct functional and cell surface properties may not belong to the long-lived, recirculating pool as defined by sensitivity to neonatal and adult thymectomy [ 8 ] . Studies of T cells that participate in the production of graft- versus-host (GVH) responses [9- 1 1 ], cytotoxic responses [8, 12-14], antigen binding [15, 161 and response to mitogens [ 171 suggest a functional heterogeneity within the T cell class based on differing homing properties, sensitivity to anti-lymph* cyte serum and to adult thymectomy. This report examines the effects of adult thymectomy upon humoral and cellular immune responses. The data indicate that the presence of the thymus in adult life is important in the regulation of helper T memory and cytotoxic responses.

[I 9591

* Supported in part by NIH grant AI-12184-01

Correspondence: Elizabeth Simpson, Division of Surgical Sciences, Clinical Research Centre, Watford Road, Harrow, Middlesex, England

2.1. Animals

CBA, BALB/cN and C57BL/6 mice aged 4-8 weeks were obtained from the Animal Production Units, National Insti- tutes of Health, Bethesda, Md. and National Institute of Medical Research, London. In some experiments, litters of 1-4-day-old (CBA x C57BL/6)F1 neonates were used.

2.2. Antisera

Rabbit anti-mouse thymocyte serum (ATS) was produced according to the method of Levey and Medawar [ 181, heat inactivated at 56 "C for 1 h, and absorbed four times with mouse red blood cells (RBC), 1 part packed RBC to 20parts ATS. One injection of 0.3 ml ATS produced a 90 % suppres- sion of GVH activity of spleen cells 3 days after administration. Anti-Thy-1.2 serum was produced according to a method previously described [8, 191. The sera used were cytotoxic to CBA thymocytes in the presence of guinea pig complement in dilutions up to 1 :6 12. The sera was rendered inactive at a concentration of 1:8 following absorption with CBA brain homogenate. The method of treatment of lymphoid cells with anti-Thy-1.2 serum and guinea pig complement has been described previously [ 81.

2.3. Cell transfers

Spleens or lymph nodes (brachial, axillary and inguinal) were excised from thymectomized or normal donors, teased into cell suspension and washed once in balanced salt solution (BSS) at 4 "C. Different numbers of these cells were then transferred intravenously to irradiated (650 r) CBA mice. In some cases the irradiated hosts also received spleen cells from donors immune to dinitrophenyl (DNP)-ovalbumin.

Abbreviations: T celk Thymus-derived cell G V H Graft-versus-host ATx: Adult thymectomy ATS: Anti-thymocyte serum OA: Oval- bumin PFC: Phque-forming cell RBC: Red blood cells BSS: Balanced salt solution DNP: Dinitrophenyl APP Alum-precipitated plus killed B. pertussis

2.4. Antigens

Bovine Serum albumin (BSA) (Cohn fraction V, Armour, Chicago, Ill.), and ovalbumin (OA) were used as antigens.

338 E. Simpson and H. Cantor Eur. J. Immunol. 1975.5: 337-343

Mice were immunized intraperitoneally with alum-precipitated protein plus 2 x 1 O9 killed B. pertussis organisms (APP), and challenged with an intraperitoneal injection of 100 pg protein or hapten-protein conjugate in saline. Anti-DNP and anti- BSA titers were assayed by the Farr technique [20].

2.5. In vitro sensitization and cytotoxic assays

These were carried out according t o the methods of Wunder- lich and Canty [ 2 1 ] and Canty and Wunderlich [ 221. In brief, 20 x 1 O6 aliquots of spleen cell suspensions t o be sensitized (responder cells) were incubated in 35 x 10 mm petri dishes (Falcon Plastics 3005, Los Angeles, Calif.) together with 1 x 1 O6 irradiated (2500 r) stimulator cells, on a rocking platform for 5 days in a COz incubator. In some experiments 5 x l o 6 responder cells were cultured in 16 x 10 mm petri dishes (Linbro Chemicals, New Haven, Conn.) together with 5 x l o 5 stimulator cells.* The cultures t o be sensitized were set up in fortified Eagle’s medium and fed daily with a nutrient medium [23]. After 5 days, the cells (“attackers”) were harvested and their cytotoxicity against a fixed number, normally 5 x 1 O 5 of ”Cr-labeled target cells assayed, a t 3 different concentrations of attacker cells, usually 2 x lo6 , 1 x l o 6 and 0.5 x lo6 per ml. The attacking cells were incubated with the target cells in 1 ml volumes in 35 m m petri dishes (Falcon) in quadruplicate, for 4 h o n a rocking platform in a COz incubator at 37 ‘C. At the end of this time, cultures were quenched with 1 ml of cold BSS fetal calf serum (FCS) and centrifuged at 2000 rpm for 1 0 min. Then 1 ml aliquots of the supernatant of each culture were counted for ”Cr activity. The cytotoxic activity of each sensitized cell population was calculated according t o the formula.

( ’ k r released by sensitized cells) - (”Cr released by unsensitized cells) ”Cr released from 4 x frozen and thawed cells

Specific lysis = 100 x

The percent specific cytotoxicity was plotted against the number of attacker cells, and the slopes of the lines obtained for each cell suspension were compared.

In experiments in which only small numbers of cells were available for sensitization, and hence assay, assays were performed at only one concentration of attacking cells, but under normal conditions there was a linear relationship between specific cytotoxicity and the number of attacking cells. In such experiments, the results are expressed both as the specific cytotoxicity observed, and as total cytotoxic activity, where total cytotoxic activity = specific cytotoxicity x the recovery of viable cells per sensitized culture. This mode of analysis was also carried out in a previous study [ 81. In every instance the results were comparable, whether the specific or total activities were used for analysis.

2.6. Thymectomy and skin grafting

Thymectomy was performed on CBA and BALB/c mice at 6 weeks of age according t o a method described previously [8]. Skin grafting was carried out according t o the method of Billingham and Medawar [ 241.

* O’Hopp, S., Wunderlich, J.R. and Simpson, E., submitted for publication

3. Results

3.1. The effect of adult thymectomy upon the in vivo and and in vitro primary response

Although the primary response t o BSA was unchanged after adult thymectomy (Fig. l ) , the early and substantial depletion of Thy- 1.2-bearing cells in spleen [ 81 suggested that iso- lation of spleen cells shortly after thymectomy might reveal an immune deficit in this tissue that was undetected in the intact animal. To test this, spleen cells obtained from mice 3 weeks after thymectomy or sham operation were transferred t o lethally irradiated recipients one day before immunizing with BSA-APP. Spleen cells from thymectomized and sham control mice transferred similar primary responses following immunization with BSA, although the slope of the dose response curve was slightly greater in the hosts receiving cells from adult thymectomized donors (Fig. 2).

7 10 14 21 28

rn ws Figure I : The primary antibody responses of CBA mice thymectomized or sham thymectomized at 6 weeks of age and primed 4 weeks later with either 400 fig alum-precipitated BSA (0, m) or 400 pg BSA-APP (o,.). Anti-BSA titers (pglml) are shown over the month following immunization. Each group consisted of 9 to 14 mice.

“r T

I

- 1

0 10 20 30 40 50 rn Number of cells x

Figure 2 : The adoptive primary antibody response of spleen cells obtained 4 weeks after adult thymedomy (0) or sham thymectomy (0). In this experiment, different numbers of spleen cells from thymec tomized or sham thymectomized donors were transferred to syngeneic irradiated (900 r) recipients 1 h prior to priming with 400 fig BSA-APP. Peak antibody titers (Day 28) are shown; vertical bars denote limits of one standard error.

Eur. J. Immunol. 1975.5: 337-343 Subclasses of T lymphocytes 339

3.2. The in vitro priniary response

Spleen cells from thymectomized and sham control mice were cultured in vitro, 15 x 1 O6 responder cells with 0.05 ml 1 % sheep (S)RBC per dish for 4 days on a rocking platform in a COz incubator, with daily feeding [23]. The cells were then harvested and assayed against SRBC. Normal spleen cells produced 17.95 * 1.29 plaque-forming cells (PFC) per lo6 cells and spleen cells from adult thymectomized mice produced 20.77 f 2.25 PFC per l o 6 cells. These figures are not significantly different.

3.3. The ability of spleen cells from thymectomized animals t o transfer a secondary response

Mice that had been thymectomized 3-4 weeks previously were immunized with BSA-APP. At different intervals there- after spleen cells from each group were transferred intravenously t o lethally irradiated syngeneic hosts, which were challenged intraperitoneally with 100 pg BSA-saline. Spleen cells from ATx donors transferred substantially lower secondary responses (approximately 5-fold lower, P < 0.05) than did cells from sham thymectomized mice (Fig. 3). These results suggested that removal of thymus resulted mainly in a defect in the ability of spleen cells t o develop immune memory, although primary responsiveness was unimpaired. In order t o establish that the defect was indeed localized in T memory activity, and to quantitate this defect, carrier-specific helper activity, mediated by T cells [25], was then assessed. Mice were immunized 3-4 weeks after adult thymectomy and sham operation with 400 pg of BSA-APP. At various intervals after immunization different numbers of spleen cells f rom thymectomized and sham thymectomized mice were transferred t o lethally irradiated hosts, along with 50 x lo6 spleen cells obtained from mice immune t o DNP-OA. These adoptive hosts were challenged 24 h later with DNP-BSA. Since an excess of DNP-OA cells was used, the titer of anti-DNP antibody was an indication of the amount of anti-BSA helper activity transferred. Anti-BSA helper memory was reduced approximately 5-fold in the adult thymectomized group,

T

T P Sham

1 10 1W m Number of cells transferred x

Figure 3: The adoptive secondary antibody response of spleen cells from thymectomized or sham thymectomized donors. CBA mice were primed with 400 fig BSA-APP 4 weeks after adult thymectomy or sham thymectomy. Four weeks later spleen cells from these donors were transferred in different numbers into syngeneic irradiated (900 r) recipients along with a challenge of 100 fig BSA in saline. Anti-BSA activity in adoptive hosts given cells from sham operated immune donors (0) was substantially higher than activity noted in recipients of spleen cells obtained from adult thymectomized immune donors (0) . Vertical bars denote limit of one standard error.

compared with sham controls (Figs. 4a and b). This effect in T memory was fairly constant over t h e first 5 weeks following immunization, was noted as early as 10 days after priming (Fig. 4a) and persisted for a t least 8 weeks (Fig. 4b).

1 10 im 1 10 1W Number of cells transferred x Number of cells transferred x 1f6

Figure 4 : The effect of ATx on the development of helper memory activity. CBA mice were primed with BSA-APP (400 pg) 4 weeks after thymectomy or sham thymectomy was performed. Spleen cells from the 2 groups were obtained 10 days (Fig. 4a) or 35 days (Fig. 4b) after immunization and adoptively transferred in different numbers to syngeneic irradiated hosts along with 40 x lo6 DNP-OA-primed spleen cells. Anti-DNP antibody activity was measured 10 days after challenge with 100 pg of DNP-BSA in saline. Spleen cells from mice primed after adult thymedomy transferred significantly less helper activity than did sham-operated controls. Vertical bars represent the limits of 1 standard error; each point represents the mean of 4-7 experiments.

The preceding experiment suggested that adult thymectomy was followed by a decreased ability t o generate immunologic helper memory. Since ATS in small doses preferentially affects recirculating lymphocytes, the effects of this reagent were compared t o the effects of adult, thymectomy. In contrast to the effects of adult thymectomy upon the primary and secondary response, a small dose of ATS (0.25 ml) administered t o mice 3 days before transfer of spleen cells t o irradiated adoptive hosts resulted in marked reduction of the primary response t o BSA, although nearly normal secondary responses could be elicited with BSA in saline 3 months later (Table 1). This secondary response was not simply due t o the recovery with time of immune reactivity in irradiated hosts, sinceirradiated hosts restored with spleen cells from ATx mice given a siniilar dose of ATS produced neither prinlary nor secondary responses (Table 1).

3.4. The effect of adult thymectomy upon cell-mediated immunity: in vitro sensitization against allogeneic antigens

Spleen cells from normal 10-week-old male BALB/c (H-2d) mice or from BALB/c mice which had been thyniectomized 3 weeks previously were cultured in vitro with irradiated (2500 r) stimulator cells from C57BL/6 (H-2b) mice. Five days later the cultures were harvested and the cells assayed for cytotoxicity against El-4 'kr-labeled target cells. The cytotoxic response produced by immunizing across an H-2 barrier (BALB/c versus C57BL/6) is shown in Fig. 5. The normal spleen cells show considerable cytotoxic activity, but that of the spleen cells from thymectomized mice is significantly higher. This experiment represents a typical result of 11 sensi- tizations over a course of 6 months. In some experiments the response of ATx spleen cells was compared with that of both sham thymectomized and normal spleen cells. Spleen cells

340 E. Simpson and H. Cantor Eur. J. ImmunoL 1975.5: 337-343

Table 1. Effect of ATx and ATS on the primary and secondary antibody response

A. PlOtomL

A’lk - S h m ATx I

4weelu I

4 weaka I

[ inkline j

25 r Slope ATx = ,$

Slope NL =

1:l 2:1 4:1 Attacker: target cell ratio

Figure 5 : The effect of ATx on primary cytotoxic responses to H-2 incompatible alloantigens. BALB/c s leen cells were sensitized in vifro for 5 days and then assayed against Gr-labeled EL4 target cells at the attacker/target cell ratios shown. Slopes are calculated linear regression curves.

B. Results

20 m - IBI r ‘A’

Anti-BSA antihdy (udrnl) ML BALB slope = 3 63 f 0 I5

Donor Rimary Secondary $ - pretreatment response

A T S ~ ) + ATX 0.14 * 0.33 AT+) 0.19 It 0.23 7.65 +- 0.99 ATx 2.15 +- 0.95 NRSb) 2.66 It 0.81 10.16 f 1.51

- x

I BALE + ALS slope . 2 I9 f 0 39

I 1 2 1 1 1 2 1 4 1 a) 40 x lo6 spleen cells injected intravenously into syngeneic irradlated

b) 0.25 ml Attacker target cell ratio (800 r) hosts.

Attacker target cell ratio

from sham operated mice gave the same response as normals, ie. less than that of ATx mice.

In contrast, treatment of mice with small doses of ATS 1-2 days before removing their spleens for in vitro sensitization resulted in the inability t o produce cytotoxic cells, suggesting that ATS-sensitive circulating T cells are necessary for the production of a primary cytotoxic response (Fig. 6a). In another experiment designed to test whether “memory” for cytotoxic cell production also required the participation 01 the circulating T cell pool, BALB/c mice which had been grafted 4 weeks previously with C57BL/6 skin were given 0.4 ml ATS. Two days later their spleens and those from sensitized, non-ATS- treated mice were removed and an aliquot of cell suspension assayed for cytotoxicity. None was found in either group. The remainder of the spleen cell suspension was sensitized in vitro for 5 days against irradiated C57BL/6 spleen cells and then assayed for cytotoxicity. The results are shown in Fig. 6b. The spleen cells from the previously skin grafted, but other- wise untreated mice produced a very high cytotoxic response far in excess of that shown by normal, nonsensitized BALB/c spleen cells cultured at the same time and therefore suggesting a memory response [ 261.

In contrast t o this, spleen cells from primed mice that had been given a small dose of ATS produced a meagre response, . suggesting the importance of ATS-sensitive, recirculating T lymphocytes for cytotoxic “memory”. Spleen cells from mice

Figure 6 : The effect of small doses of ATS on the ability of spleen cells from mice given 0.3-0.4 ml ALS subcutaneously 48 h previously, to make a primary (A) or secondary (B) cytotoxic response to alloantigens. In vitro sensitization for 5 days was followed by assay of cytotoxicity against “Cr-labeled EL4 cells, at the attackerbarget cell ratios shown. Slopes are calculated linear regression curves.

which had been sensitized by skin grafting 3 weeks after adult thymectomy developed unimpaired memory cytotoxic responses when placed in culture 28 days after grafting (Table 2, Exp. 1) suggesting that the ATx-sensitive, non-recirculating T cell was not involved in the induction of memory cells for the cytotoxic response.

The spleen cells sensitized in virro in the above experiments were coniposed of both B and T cells. The cytotoxic response is a function of T cells, although small numbers of macrophages are needed during the sensitization phase in vitro [26, 271. In order t o test whether the enhanced responsiveness of spleen cells from ATx mice and the expression of memory for the cytotoxic response were T cell functions, nylon wool columns were used t o prepare purified T cell populations from spleens before sensitization [27]. The nylon wool removes both B cells and macrophages [27], SO it was necessary t o add back small numbers of peritoneal wash cells as a source of macrophages t o t h e cultures (2 .5 x 10’ peritoneal wash cells to 5 x I O b responders) during sensitization. For these experiments 16 x 10 mm petri dishes (Linbro) were used in order t o reduce the number of purified splenic T lymphocytes used.

. -

Eur. J. Immunol. 1975.5: 337-343 Subclasses of T lymphocytes 341

Table 2. Cytotoxic memory responses: effect of ATx and dependence on T cells

Source of ap~leen OW)

Exp. 1

cytotoxic activity“)

1.45 f 0.38 2.18 f 0.32 4.03 f 0.22

Nonimmune, no Nonimmune, ATxc ~mmuned). n o d m u n e d ) ATXC) 6.21 f 0.53

Exp. 2

Nonimmu~ 16.33 f 0.34 Immune ‘T” 34.27 f 2.19

T1

Balb/c responder spleen cells were sensitized in vitro with C57BL/6 antigens for 5 days prior to assay. Cytotoxic activity against ’’Cr-labeled EL4 target cells is given as slope of the titration curves, as shown in Figs, 5 and 6, using attacker/target ratios of 1:l to 4: l . Adult thymectomy performed three weeks prior to skin grafting, i.e. 7 weeks prior to in vitro sensitization. By grafting with C57BL/6 tail skin 4 weeks prior to sacrifice. Prepared by use of nylon volumns [29].

The results of 3 such experiments are shown in Table 3. Clearly the enhanced responsiveness of T cells from spleens of mice thymectomized 3 weeks previously in comparison with those of normal mice is still apparent, suggesting that t h e increased responsiveness of spleen cells following ATx reflects an altered T cell response. Likewise, memory for the cytotoxic response also resides in T cells (Table 2, Exp. 2).

Table 3. Generation in vitro of cytotoxic activity by purified splenic T cells: effect of ATx

Source of splenic T cell&

Exp. 1 Normal

Exp. 2 Normal

Exp. 3 Normal

ATXC)

ATx

ATx

Cytotoxic activityb)

1.60 f 0.14 5.10 f0.13 7.20 f 0.5 1 16.10 f 0.40 6.64 * 0.43 18.19 f 1.37

a) Splenic T cells from BALB/c mice prepared by use of nylon wool columns [29] then used as responder cells, sensitized in vitro to C57BL/6 antigens for 5 days prior to assay.

b) Cytotoxic activity, as in Table 2. c) Adult thymectomy performed 3 weeks prior to in vitro sensitization.

In order t o establish a dose-response curve for sensitization of T cells from ATx and normal mice, graded doses of cells from the two groups were sensitized over 5 days. Each well was made up t o 5 x lo6 cells, using anti-theta-treated “filler” cells. These experiments showed that the main effect of ATx was t o permit substantially higher levels of sensitization when relatively large numbers of cells were used (Table 4).

3.5. Cytotoxic activity generated by combination of splenic T cells from ATx and ATS-treated donors

The altered capacity of T cells from adult thymectomized donors to generate primary cytotoxic responses following in v i t ro sensitization could be accounted for by the idea that adult thymectomy removes a population of T cells that exert

Table 4. The cytotoxic response of splenic T cells from normal (NL) and adult thymectomized (ATx) mice: effect of varying the number of responder T cells per well

ATx-T NLT [x 106) TAI) (x 106) TA

1 4.5 f 0.09 1 6.3 f 0.15 2.5 16.4 f 0.40 2s 10.9 f 0.20 5 39.1 f 0.91 5 16.3 f 0.21

a) Total lytic activity (TA) generated following sensitization of different numbers of spleen T cells obtained from ATx and normal mice. The total numbers of cells sensitized were kept constant at 5 x lo6 by the addition of appropriate numbers of spleen cells following treatment with anti-theta plus complement.

a regulatory role upon in v i t ro induction of cytotoxic activity. In order t o test this idea, nylon-passed spleen cells from ATx mice were mixed with different numbers of nylon passed cells from the spleens of mice that had been given a small dose of ATS 2 days prior t o sacrifice ( “SPA-T”). SPA-T cells produced negligible levels of lytic activity after sensitization alone, despite the fact that the proportions of viable T cells in these nylon- passed preparations were similar t o normal column-passed spleen cells both before and after in v i t ro sensitization [ 141. The addition of SPA-T t o small numbers of spleen cells from ATx mice resulted in enhanced development of cytotoxicity (Fig. 7). However, the addition of SPA-T t o relatively larger numbers of spleen cells from ATx mice (ATx : ATS = 4: 1) resulted in slight suppressive effects. In order t o determine whether larger numbers of SPA-T cells could suppress a vigorous response, 4 x 1 O6 spleen cells from ATS-treated mice were added t o 16 x 1 O6 spleen cells from ATx donors. The addition of ATS-spleen cells substantially suppressed the response (Table 5 , Exps. 1-3); this suppressive effect was abrogated by pretreatment of ATS- spleen with anti-theta plus complement (Table 5, Exp. 3). These experiments indicate that ATx results in impairment in the function of splenic, ATS-resistant T cells that suppress the development of cytotoxicity during a vigorous response. When, however, in similar experiments the response was rather low, the presence of ATx-sensitive T cells resulted in amplified reactivity, both of ATx and normal spleen cells (Table 6, Exps. 1-3).

- ATx-T 0 I 2 3 4 S ATs-T 5 4 3 2 I 0

Figure 7 : Effect of mixing together various proportions of splenic T cells from ATx and ATS-treated mice, prior to in uirro sensitization, on the cytotoxic response to alloantigens. ATx mice were thymec tomized 3 weeks prior to use: mice given ATS received 0.3 ml s/c 48 h prior to use of spleens. Figure shows mean * SEM of 4 experk ments. - gives specific activity of cell suspension assayed, O-----O

gives cytotoxic units per culture, i.e. specific activity x cell recovery per culture. A total of 5 x lo6 per well were cultured during sensitization (Linbro plates, see Section 2.5.).

342 E. Simpson and H. Cantor Eur. J. Immunol. 1975. 5 : 337-343

Table 5. Effect of adding back ATS-resistant spleen cells to in vitro culturesa) of spleen cells from ATX mice

ATx NL ATS % lysisb)

Exp. 1

20 - - 10.86 f 1.13 16 - - 10.31 f 0.69

20 - 2.25 f 0.08 - 16 - 2.73 f 0.07 16 - 4 3.33 f 0.40

-

Exp. 2

20 - - 12.59 f 0.75 16 - - 10.83 f 1.22 - 20 - 10.72 f 0.50 - 16 - 8.59 f 1.11

16 - 4 8.55 f 0.48

Jkp. 3 20 - - 15.11 f 1.11 16 - - 13.80 f 2.53 16 - 4c) 9.72 f 2.10 16 - 4d) 13.90 f 1.14

a) BALB/c responder spleen cells were sensitized in vitro in 35 mm petri dishes with C57BL/6 antigens for 5 days prior to assay.

b) Cytotoxic activity against 5vr-labeled E L 4 target cells is given as slope of titration curves, as shown in Figs. 5 and 6 , using attacker/ target ratios of 1 : 1 to 4: 1.

c) Brain-absorbed an t i4 serum + C. d) Anti4 serum + C.

Table 6. Effect of addin back ALS-resistant spleen cells to ATx or N L in vitro spleen culturesaf’rnaking relatively low cytotoxic responses

ATx NL ATS I lysisb)

Exp. 1 - 8.02 f 0.34 20 -

16 - - 4.51 f 0.53 16 - 4 7.56 f 0.84

Exp. 2 20 - - 1.88 f 0.07 16 - - 1.27 * 0.16 16 - 4 4.90 f 0.15

Exp. 3 0.73 f 0.16

16 - 0.68 f 0.12 16 4 1.95 f 0.09

- 20 - - -

a) BALB/c responder spleen cells were sensitized in vitro in 35 mm petri dishes with C57BL/6 antigens for 5 days prior to assay.

b) Cytotoxic activity against ”Cr-labeled EL4 target cells is given as slope of titration curves, as shown in Figs. 5 and 6, using attacker/ target ratiosof 1 : l to 4.1.

4. Discussion

These experiments suggest that the thymus continues t o play an important role in regulating the immune response in adult life. Shortly after adult thymectomy, animals develop enhanced primary killer cell responses to alloantigens as well as diminished helper memory responses. These findings extend those reported previously [28, 291 and indicate that the thymus can regulate both cellular and antibody responses in adult life. Loss of this regulatory mechanism may be reflected in other early conse-

quences of adult thymectomy such as increased mixed lympho- cyte culture reactivity [30] and increased primary antibody responses t o certain antigens [3 1 , 321.

Although these findings indicate that the thymus can regulate both the type and strength of an immune response, they d o not directly establish the cellular mechanism underpinning this control. Previous studies have indicated that within one month after adult thymectomy (a) the proportion of Thy-]+ cells in spleen is decreased by 30-50 % [8, 291 and (b) there is a marked decrease in a subclass of Thy-]+ spleen cells that form rosettes with sheep erythrocytes (T-RFC) [33]. The latter subclasses of T-RFC, sensitive t o very low concentrations of anti-Thy-I, can be restored following administration of thymic extracts or factors [33]. The present studies indicate that these cells, designated T-l [29] can (a) contribute t o the generation of T memory cells, after priming with antigen, and (b) suppress the development of killer cells, especially when relatively large numbers of cells are responding (Table 4). I t is not clear whether these ATx-sensitive cells account for all of the regulatory activity (amplifier and suppressor) described in the preceding paper [ 141 or are limited t o suppressing killer responses.

The altered immune responses noted after adult thymectomy as described here and elsewhere [ 28-32] have similarities with the immunologic abnormalities that characterize several inbred mouse strains, including NZB and HRS/J. NZB mice are resistant t o tolerance induction [ 341, develop substantial numbers of T cells which form rosettes with syngeneic RBC [35] and demonstrate abnormally vigorous and prolonged proliferative responses to alloantigens [36]. The latter two findings are a t least partially corrected by thymic factors or extracts [36]. HRS/J mice undergo severe thymic cortical atrophy at 5-6 months of age, associated with an inability t o develop signifi- cant secondary antibody responses t o tetanus toxoid, despite near-normal primary responses [37]. It is possible that the autoimmune disorder and high incidence of neoplasia in these strains may reflect, in part, the immunologic consequences of “autothymectomy” in adult life.

Memory for cytotoxic responses is not nearly so well charac- terized as memory for humoral responses, where memory activity has been shown t o reside in both T and B lymphocytes. The present studies show that T cells from mice previously primed by skin grafting and then sensitized or “boosted” in v i t r o t o the same alloantigens produce substantially higher levels of cytotoxic activity than those generated by T cells from virgin, nongrafted mice. Since T cells from skin grafted mice demonstrated negligible levels of cytotoxicity before in v i t r o “boosting” [ 261, this experiment demonstrates that cytotoxic memory is carried in T cells. Further experiments indicated that cytotoxic memory is carried by the ATS-sensitive cell subset.

We wish to thank Ms. Susan O’Hopp, Ms. Joanne Hugenberger and Ms. Marianne Watson for their skillful assistance.

Received November 15, 1974.

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5 Taylor, R.B., Nature 1965.208: 1334. and Papermaster, B.W., J. Exp. Med. 1962. 1 1 6 : 773.

Eur. J. Immunol. 1975. 5: 343-349 Antiphosphorylcholine response in microcultures 343

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2018.

H. Cosenza, J. Quintans and 1. Lefkovits

Basel Institute for Immunology, Basel

Antibody response to phosphorylcholine in v i m

1. Studies on the frequency of precursor cells, average clone size and cellular cooperation

The anti-phosphorylcholine (PC) antibodies synthesized by BALB/c spleen cells in microcultures upon immunization with heat-killed vaccine of Pneumo- cocci R36A (Pn) are directed exclusively t o the PC epitope. These antibodies are of very restricted avidity and 88 % of the responding clones express the idiotype characteristic of the TEPC-15 PC-binding myeloma. This idiotypic restriction appears t o be due t o the absence of clones capable of expressing other idiotypes, rather than t o “clonal dominance”.

The estimated frequency of precursor cells for the PC epitope is 1 x lo-’ t o 2.5 x 1 O-’. These precursors give rise to clones with an average size of 9 plaque-forming cells. When the logarithm of the number of negative wells was plotted against the number of spleen cells/well, the fraction of nonrespond- ing cultures decreased exponentially as the number of spleen cells was increased. This indicated that only one cell type was limiting in our assay, presumably a B cell. Furthermore, treatment of spleen cells with AKR anti-0 serum completely abolished the response t o sheep red cells without affecting the response t o PC. It is concluded that PC is a T cell-independent antigen. Of interest was the finding that PC requires adherent (A) cells and this is a particular characteristic of PC, since most T cell-independent antigens have been found not t o require A cells. Reasons for the possible homogeneity of the response t o PC are also discussed.

[I 10211

Correspondence: Humberto Cosenza, Basel Institute for Immunology, 487, Grenzacherstrasse, CH-4058 Basel, Switzerland

Abbreviations: PC: Phosphorylcholine PnC: C-polysaccharide from pneumococci R36A pn: Heat killed vaccine o~pneumococci R36A SRC: Sheep red cells PnC-SRC: SRC coated with PnC 2-ME: 2-Mer- captoethanol BSS: Hanks’ balanced salt solution PFC: Plaque forming cell(S) T Cell: Thymus-derived ceU(S) B Cell: Bone marrow- derived cell(s) A cell: Adherent (macrophage-like) cell(s) FCS: Fetal calf Serum GPC: Guinea pig complement TNP-LPS: 2,4,6-Trinitro- phenyl coupled onto bacterial ljpopolysacchdde DW: 2,4-Dinitro- phenyi NMS: Normal mouse serum MEM: Minimum essential medium

1. Introduction

The antibodies produced after antigenic stimulation comprise molecules with a wide range of avidities and only after a prolonged maturation of the response does a narrower range of more avid antibodies arise [ 1, 21. This antibody heterogeneity is thought to reflect the broad repertobe of precursor cells which can interact with a particular antigen and the fact that new specificities might be generated within proliferating clones during the course of an immune response [3].

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