Immunology 1977 33 757

Thymic-dependent anti-hapten response in congenitally athymic (nude) mice inmunized with DNP-thymosin A. AHMED*, A. H. SMITH*, K. W. SELL*, M. E. GERSHWINt, A. D. STEINBERG t, G. B. THUR MAN §, & A. L. GOLD STEIN § Cellular Immunology Division, Department of Clinical and Experimental Immunology, Naval Medical Research Institute, Bethesda, Maryland 20014, tSection of Rheumatology, Department of Internal Medicine, School of Medicine, University of California, Davis, California 95616, tArthritis and Rheumatism Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014, §Division of Biochemistry, University of Texas Medical Branch, Galveston, Texas 77556

Received 17 February 1977; accepted for nublication 17 March 1977

Summary. Immunization of congenitally athymic (nu/nu) and adult thymectomized, irradiated bone marrow, reconstituted (TxBm) mice with DNP5thymosin (dinitrophenylated-bovine thymosin fraction 5) was found to elicit IgM and IgG anti-DNP plaque-forming cells in these animals. Further studies indicated that this response was antigen specific and not due to polyclonal activation. Since the hormonal properties of the thymosin were retained following linkage with hapten and DNPthymosin was immunogenic in CBA/N and (CBA/N V x DBA/2 ,)Fl d mice, animals previously shown to have an X-linked inability to respond to thymusindependent antigens, it was concluded that DNPthymosin functions both as a hormone and as a T-dependent antigen in eliciting an immune response in nu/nu and TxBm mice. Additional support for this conclusion was provided by the demonstration that DNP-thymosin could specifically prime for and elicit an anamnestic response in nu/nu mice. These results indicate that further investigation of the immune activities of DNP-thymosin may provide

valuable insight in characterizing the maturation of helper T cells and their subsequent interaction with B cells. INTRODUCTION The importance of the thymus in the maturation of lymphoid cells, particularly with relevance to its role in the humoral response to a wide variety of antigens, has been well documented (Miller, 1971). The thymus-derived (T) cells that are necessary for the initiation of B-cell responses to certain antigens have been classified helper 'T' cells and are a distinct subpopulation of T cells. Thus, using neonatal thymectomy followed by replacement with thymocytes, the role of helper T cells in the induction of the antibody response to thymic-dependent antigens has been well defined (Miller & Mitchell, 1968). The mechanism for helper T-cell function was interpreted as either secondary to lymphoid cell traffic through the thymus or due to humoral factor(s) produced by the thymic tissue. The role of the thymic humoral factors in the maturation of T cells was further strengthened by the results of implantation of thymic tissue in diffusion chambers in both

Correspondence: Dr Aftab Ahmed, Clinical and Experimental Immunology Department, Mail Stop 24, Naval Medical Research Institute, Bethesda, Maryland 20014, U.S.A.

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neonatally thymectomized experimental animals (Miller & Osoba, 1967) and children with the DiGeorge syndrome (Steele, Limas, Thurman, Schuelein, Bauer & Bellanti, 1972). With the discovery and usage of congenitally athymic nu/nu mice, it was subsequently shown (Gershwin, Ahmed, Steinberg, Thurman & Goldstein, 1974) that administration of bovine thymosin 5 (a mixture of polypeptides prepared from thymus tissue) partially corrects the helper T-cell defect in the response of mice to sheep red blood cells (SRBC) and dramatically increases lymph node cell responses to concanavalin A (Con A) (Thurman, Ahmed, Strong, Gershwin, Steinberg & Goldstein, 1975). It was the purpose of this study to determine whether the administration of dinitrophenyl (DNP) linked to thymosin fraction 5 would lead to both the maturation of precursor helper T cells and the concurrent development of antibody to the hapten in thymectomized mice and athymic nu/nu mice. We report herein that hapten linked to thymosin does not alter its ability to act as an inducer for the amplification of T-cell function. Moreover, DNP-thymosin induces specific T-cell helper function. These studies illustrate a unique model for exploration of T-cell maturation and the mechanism of T and B cell co-operation for an anti-hapten specific response.

MATERIALS AND METHODS Mice

Congenitally athymic nu/nu mice on an N: NIH(S) background and their normal littermate + /nu controls, CBA/N and the (CBA/N Y x DBA/2 S)Fj male and female mice, were obtained from the Rodent and Rabbit Production Unit of the National Institutes of Health, Bethesda, Maryland. BALB/c and C3H/He mice were obtained from the Jackson Laboratories, Bar Harbor, Maine. Eight-week-old mice of both sexes were used in these studies. Thymosin fraction 5 Thymosin was prepared from calf thymus tissue as described previously (Hooper, McDaniel, Thurman, Cohen, Schulof & Goldstein, 1975), and fraction 5 was used in these studies.

DNP5-thymosin Thymosin fraction 5 was coupled with DNP (Little & Eisen, 1967) and the product was dialyzed

against 0-15 M saline using an Amicon UM-2 membrane filter. The average number of DNP groups per mole of thymosin was determined as described (Little & Eisen, 1967), based on an average molecular weight of 8000 daltons for the polypeptides in thymosin fraction 5. A large batch of this DNP5-thymosin was prepared and stored in aliquots at -20° until use. DNP32-lys-Ficoll, previously characterized to be a thymus-independent antigen, was a generous gift of Dr W. E. Vannier. SRBC were obtained from Microbiological Associates, Bethesda, Maryland, and washed three times before use. The same sheep was used as a donor of SRBC throughout the study. Cell suspensions Spleen and lymph node cell suspensions were prepared as described previously (Ahmed, Scher & Sell, 1977).

Plaque-forming assay The spleen suspensions from saline control and immunized mice were evaluated for their ability to form both IgM and IgG plaques by a slide modification of the Jerne plaque assay (Scher, Steinberg, Berning & Paul, 1975). The number of IgG-forming cells was determined by the addition of anti-p to suppress the formation of IgM plaques, followed by development with anti-mouse IgG. Response of nu/nu and + /nu mice to DNP-thymosin Groups of nu/nu and + /nu mice were injected with either saline or 1, 10, 50, 100, or 250 pg of DNPthymosin i.p. in a volume of 0 5 ml. Four and 7 days later the mice were sacrificed by cervical dislocation and their spleens aseptically removed for quantification of both direct and indirect splenic plaqueforming cells. Each group consisted of at least four nu/nu and four + /nu mice for each dose of antigen per day of assay.

Secondary response of + /nu and nu/nu mice to DNPthymosin Groups of 24 nu/nu and + /nu mice each were primed with either saline, 100 ug DNP-thymosin, or 100 ug DNP-OVA and, subsequently, eight mice in each group were challenged 2 weeks later with either saline, 10,pg of DNP-thymosin, or 10 pg of DNPOVA. Three and 5 days after secondary challenge, four mice from each subgroup were sacrificed and

Anti-hapten response in congenitally athymic mice their spleens assayed for both IgM and IgG-PFC using TNP-burro red blood cells (RBC).

Specificity of the TNP response of nu/nu and + nu mice to DNP-thymosin Groups of four nu/nu and four +/nu mice were immunized with either saline, 10, 100, or 250 pg of DNP-thymosin in a volume of 0 5 ml i.p. The mice were sacrificed 4 days later and the direct PFC response of spleen cells was determined to SRBC (nonspecific polyclonal) and TNP-burro RBC

(specific).

Effect of DNP-thymosin and thymosin fraction 5 on the mitogen response During each of the assays above, an aliquot of spleen cells was prepared for mitogen assays. Similarly, lymph node cell suspensions from both axillary and mesenteric lymph nodes were prepared. These lymphocyte suspensions were then cultured with the T-cell mitogens phytohemagglutinin-P (PHA-P), Con A, and the B-cell mitogens bacterial lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I: poly C), as previously described (Strong, Ahmed, Thurman & Sell, 1973). Response of TxBm and CBA/N mice to DNP-thymosin Adult BALB/c mice were thymectomized, irradiated, and 1 day thereafter reconstituted with anti-thy 1-2 and complement-treated, syngenic bone marrow cells. An equal number of control mice, consisting of sham thymectomized mice, were included. Two weeks later, groups of six control and six TxBm mice were immunized with 100 pg of DNP-thymosin, 0-2 ml of 10%Y SRBC, or 50jpg DNP-lys-Ficoll. Additional control mice, consisting of both sham thymectomized and TxBm animals, were injected with 0-5 ml of saline. Four days later their spleens were assayed for IgM-PFC, using SRBC and TNPburro RBC. CBA/N and (CBA/N ? x DBA/2 d)F1 mice were used to evaluate the thymus-dependence of the response to DNP-thymosin. These mice have previously been shown to have an X-linked B lymphocyte immune defect (Amsbaugh, Hansen, Prescott, Stashak, Barthold & Baker, 1972), and CBA/N and F1 male mice do not respond to thymusindependent antigens such as DNP-lys-Ficoll, whereas F1 female littermates are normal in their response to these antigens (Scher et al., 1975). Thus, if the response of these mice to DNP-thymosin was

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similarly controlled in an X-linked fashion, it would be strong evidence that DNP-thymosin functions as a typical thymus-independent antigen, and does not require helper T cells to initiate an immune response. RESULTS Response of + /nu and nu/nu mice to DNP-thymosin As seen in Table 1, administration of 50 ug of DNPthymosin induced significant direct PFC in spleens of + /nu mice (641 ± 26 D-PFC/spleen). In contrast, nu/nu mice responded to as little as 10 pg of DNPthymosin (881 ± 365 D-PFC/spleen). Indeed, at doses of 10, 50, and 100 ug of DNP-thymosin, the response of the nu/nu mice was statistically higher than that of + /nu mice (P< 0 01). At doses of 250 ug of DNP-thymosin, the IgM response was similar in both groups. Both + /nu and nu/nu mice also exhibited a significant IgG response to DNPthymosin, as measured on day 7. As seen in Table 1, as little as 10 ug of DNP-thymosin induces a significant 7S response in both +/nu (497±56 I-PFC/ spleen) and nu/nu (668 ± 234 I-PFC/spleen) mice. Although at larger doses of DNP-thymosin the response of nu/nu mice exceeded that of the + /nu, they both exhibited similar responses. It is clear from these data that both + /nu and nu/nu respond well to a primary immunization with 100 pg of DNP-thymosin by the production of both IgM and IgG plaques. Kinetics of the response of +/nu and nu/nu mice to 100 ,ug of DNP-thymosin A distinct IgM response to DNP-thymosin was observed as early as day 2 after immunization of both + /nu and nu/nu mice with 100,pg of DNPthymosin (Fig. 1). The peak IgM response occurred on day 4-5 and this response gradually decreased to background levels by day 7. On the other hand, a significant IgG response was observed on days 4-5 and the peak IgG response for both + /nu and nu/nu mice occurred on days 7-8 after immunization with 100 pg of DNP-thymosin. These data represent a mean of four + /nu and four nu/nu mice per day and indicate optimum time points for the observation of IgM and IgG-PFC.

Specificity of the TNP response of +/nu and nu/nu mice to DNP-thymosin

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+1

60 Sain

3

225

E 0 C

U-

b40 -15-

~0 E z

to~~~~~~~~~~~~~D ys potimnnigio 20 -7-5-

Saline 2 control

3

4

5

6

Saline 4 control Days post immunizat ion 7

5

6

7

8

9

10

Figure 1. Kinetics of the TNP response of +/nu (open columns) and nu/nu (hatched columns) immunized with 100,ug DNP,thymosin i.p. At daily intervals a different group of four + /nu and four nu/nu mice was immunized with DNP,-thymosin, and 2 days following the last injection all mice were sacrificed and their spleens assayed for direct (a) and indirect (b) plaqueforming cells.

Table 1. Anti-hapten IgM and IgG response of + /nu and nu/nu mice to various concentrations of DNP-thymosin* Number of TNP plaques Dose of DNP-

Mean D-PFC/spleen + s.e.t

Mean I-PFC/spleen ± s.e.t

thymosin (Ug) 1 10 50 100 250

+ /nu

nu/nu

+ /nu

nu/nu

129±42 305±112 118±68 87±57 263 ±101 475 ±184 200 ±51 103 ±29 231 ±49 668 ±234 881 ±365 497 ±56 987 ±69 641 ±26 1168 ±194 683 ±49 1012±283 2387±324 1381 ±298 1586±136 2281 ±263 2743 ±406 1224 ±304 1339 ±247

* Groups of +/nu and nu/nu mice were injected i.p. with 1, 10, 50, 100, or 250 pg of DNP-thymosin in 0-5 ml each. Controls received saline. t The mean number of IgM (D-PFC/spleen) of individual mice was determined on day 4 and the data represent the mean of mean ± s.e. of four mice run in duplicate. $ The mean number of IgG (I-PFC/spleen) of individual mice was determined on day 7 and the data represents the mean of means ± s.e. of four mice run in duplicate.

In order to determine if the response of +/nu and nu/nu mice to DNP-thymosin was specific for the hapten, groups of these mice were immunized with various doses of DNP-thymosin and their spleen cells evaluated for PFC using SRBC (nonspecific) and TNP-burro RBC (specific) as target antigens. As seen in Table 2, at the various doses tested, while there was a distinct response by both + /nu and nu/nu mice, as measured by TNP-burro RBC (e.g. 4833± 1263 and 7116± 1148 D-PFC/spleen with 100 pg of DNP-thymosin) there was virtually background PFC with SRBC. These data suggest the response to the hapten was antigen specific and not due to polyclonal stimulation. Moreover, in separate experiments (data not shown), when both + /nu and nu/nu mice were immunized with carrier alone (thymosin), no response was noted against either SRBC or TNP-burro RBC. In order to determine if there was any alteration in the hormonal activity of thymosin secondary to its

linkage with DNP, in each experiment regional lymph node cells were obtained and cultured in vitro

Anti-hapten

response

Table 2. Specificity of the TNP response of + /nu and nu/nu mice immunized with DNP-thymosin*

sint (pg) -

10 50 100 250

Table 4. Primary IgM response of sham thymectomized and TxBm mice to DNP-thymosin, DNP-lys-Ficoll and SRBC

Mean D-PFC/spleen ± s.e4

Dose of DNPthymo-

Group of mice*

TNP-burro

SRBC + /nu

nu/nu

+ /nu

nu/nu

308 ±79 233 ±121 194 ±58 233 ±106 183 ±36

183 ±88 258 ±65 237 ±101 583 ±158 262 ±137

1408 ±375 3176 ±593 2887 ±412 4833 ±1263 4266 ±824

1166 ±346 3966 ±636 5538 ±601 7116 ±1148 9250 ±542

* Response of spleen and lymph node cells from each mouse to the T-cell mitogens PHA-P and Con A and the B-cell mitogens LPS and Poly I.C. was evaluated as a measure of the efficiency of thymosin administration (see Table 3). f Each group of four mice was immunized i.p. with 10, 50, 100, or 250 pg of DNP-thymosin in a volume of 0 5 ml, Controls received 0 5 ml of saline. $ Mean IgM (D-PFC) response was the mean of means of data from four mice/group performed in duplicate.

with Con A, as previously described (Thurman et al., 1975). The results of a typical experiment are shown in Table 3. As seen, there is a marked enhancement in the responsiveness of lymph node cells from thymosin or DNP-thymosin-treated mice to Con A. These data suggest that hapten linkage of thymosin Table 3. Lymph node cell response to Con A of +/nu and nu/nu mice immunized with thymosin or DNP-thymosin

Mice immunized with*

Saline Saline

Thymosin Thymosin DNP-thymosin DNP-thymosin

Cultured in vitro witht

Mean uptake of [3H1-thymidine ± s.e.t + /nu

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in congenitally athymic mice

nu/nu

341 ±39 128 ± 19 586 ±241 3887 ±196 498 ±54 337 ±48 Con A 111,264 ± 1458 118,918 ± 2454 595 ± 78 446 ± 51 Con A 135,563 ± 2489 129,933 ± 3456

Con A

* Groups of six mice were immunized with 100 pg of thymosin or DNP-thymosin every-other day for a total of 400 pg. One day after the last immunization their regional lymph node cells were assayed individually for their ability to respond to Con A. t Cultures received varying concentrations of Con A and only peak responses are reported here. The peak ranged between 0 03 and 0 25 pg of Con A/culture. $ Cultures were performed in triplicate and the mean uptake of [3H]-TdR determined on day 3.

Sham Tx TxBm Sham Tx TxBm Sham Tx TxBm Sham Tx TxBm

Immunized

witht Saline Saline SRBC SRBC

Mean D-PFC/ spleen ± s.e.$ 436 ± 181 589 ± 226 34,418 ± 5748 1784 ± 642

DNP-lys-Ficoll 72,433 ± 12,491 DNP-lys-Ficoll 29,840 ± 6461 DNP-thymosin 10,218 ± 2374 7481 ± 1267 DNP-thymosin

Mean D-PFC/106 cells ± s.e.

3-14 ±1-09 6-47 ± 1-83 246 ±43 26 ± 5 384 ± 68 410 ± 83 126 ± 19 61 ± 27

* Each group consisted of six mice. t Mice were immunized i.p. with 0-5 ml containing either saline, 50 pg of DNP-lys-Ficoll, or 100 pg of DNP-thymosin, or with 0 5 ml of a 10 per cent SRBC suspension. T Primary IgM response was measured on day 4 against SRBC for mice immunized with SRBC and TNP-burro RBC for mice immunized with DNP-lys-Ficoll and DNP-thymosin.

does not alter its ability to act as a hormone, at least as measured by the enhanced response of regional lymph node cells to Con A. Response of T- and B-cell-depleted mice Since it was established that nu/nu mice respond to DNP-thymosin, and this response was specific for DNP, further experiments were carried out using TxBm mice and CBA/N mice, a strain of mice possessing an X-linked B-lymphocyte immune defect. Besides DNP-thymosin, two other antigens were used for control purposes. These included SRBC for a T-cell-dependent antigen and DNP-lysFicoll for a T-independent antigen. As seen in Table 4, sham thymectomized mice responded well to all three antigens. In marked contrast, TxBm mice responded very poorly to SRBC, but responded well to DNP-lys-Ficoll (410± 83 D-PFC/106 as compared to 384±68 D-PFC/106 for sham thymectomized) and DNP-thymosin. These data do not rule out the possibility that DNP-thymosin could be a Tindependent antigen. Therefore, use was made of the inbred CBA/N and DBA/2 mice and the F1 mice derived from the matings of these two strains, and the response of these mice to the three antigens, SRBC, DNP-lys-Ficoll, and DNP-thymosin, evaluated. As seen in Table 5, DBA/2 and the

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Table 5. Primary response of CBA/N and (CBA/N V x DBA/2 6)F1 mice to DNP-

thymosin Mean D-PFC/spleen ± s-e-t

Strain of mice*

Saline

DNP-lys-Ficoll

CBA/N DBA/2 F1 S F1 V

358 ± 46 537 ±79 236±39 586±101

SRBC

DNP-thymosin

289 ± 52

12,742 ± 1029

48,489 +5051

27,433 +2419

16,729 ± 1739 19,941 ± 3490 10,770±639 29,795 ±2467

189 ±24

15,413 ±1376

56,473 +6814

37,896+4889

* Each group consisted of six mice and each group of mice received either 100 gg DNP-thymosin, 50 pg of DNP-lys-Ficoll, or 0 5 ml of 10 per cent SRBC. Controls received saline. t Mean IgM (D-PFC) response of spleen cells from each individual mouse was determined using SRBC for SRBC-immunized mice and TNP-burro RBC for DNP-thymosin- and DNP-lys-Ficoll-immunized mice. Each value represents the mean of means of data obtained from the spleens of six mice assayed individually. The plaque assay was performed in duplicate.

(CBA/N Y x DBA/2 S)F1 female mice responded well to all three antigens tested. In marked contrast, CBA/N mice and the (CBA/N Y x DBA/2 6)F1 male mice did not respond at all to DNP-lys-Ficoll, as expected, but did respond appreciably to both SRBC and DNP-thymosin. Although these data strongly suggest that in mice DNP-thymosin is not a Tindependent antigen, they do not rule out the possibility that DNP-thymosin stimulates primitive B cells present in CBA/N or F1 mice derived from this strain in a T-independent fashion. Secondary IgM and IgG response of + /nu and nu/nu mice to DNP-thymosin Thymus-independent antigens, such as DNP-lysFicoll, SIII, etc., do not give rise to a secondary response (Basten & Howard, 1973). Experiments were therefore carried out to determine if immunization with DNP-thymosin would give rise to a secondary response. A preliminary experiment was carried out to determine the optimum dose for primary and secondary challenge and the optimum time for both IgM- and IgG-PFC. It was found that primary challenge with 100jpg of DNP-ovalbumin (DNPOVA) or DNP-thymosin, followed 2 weeks later with 10 ,ug of homologous hapten carrier, gave peak IgM-PFC on day 3 and peak IgG-PFC on day 5. The experimental protocol is described in the Materials and Methods section. As seen in Table 6, both + /nu and nu/nu mice preimmunized with DNP-thymosin gave a significant secondary IgM

response when challenged with the homologous hapten carrier (18562 ± 1225 for + /nu and 8231 + 1381 D-PFC/spleen for nu/nu). It is of interest that +/nu mice gave a better secondary IgM response than nu/nu mice. On the other hand, only + /nu mice preimmunized with DNP-OVA gave a secondary response upon challenge with DNP-OVA, but not nu/nu mice. The response was specific in that DNP-OVA-preimmunized mice challenged with DNP-thymosin showed no secondary response in both +/nu and nu/nu mice. Also, DNP-thymosinpreimmunized mice did not develop a secondary anti-DNP response upon subsequent challenge with DNP-OVA. Similar results were obtained with the IgG response on day 5. As seen in Table 6, both +/nu and nu/nu mice preimmunized with DNPthymosin gave a very significant secondary IgG response when challenged with a secondary dose of 10,ug of DNP-thymosin. Again, only +/nu mice gave a secondary to DNP-OVA, demonstrating the need for helper T cells and carrier-specific memory cells for secondary response to DNP-OVA.

DISCUSSION The concept that thymic factors can influence the differentiation and maturation of T cells led numerous investigators to the extraction, purification, and characterization of products from the thymus that possess biological activity. These have included preparations such as thymosin (Goldstein,

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Anti-hapten response in congenitally athymic mice Table 6. Secondary IgM (D-PFC) and IgG (I-PFC) response of +/nu and nu/nu mice to DNP-thymosin

Primary immunization with*

Secondary immunization

witht

Mean D-PFC/spleen (mean + s.e.)t + /nu nu/nu

Mean I-PFC/spleen (mean ± s.e.)§ + /nu nu/nu

Saline

Saline 10Opg DNP-OVA 10pg DNP-thymosin 100ug DNP-OVA 100 pg DNP-thymosin

200±66 346 ±91 795 ± 312 647 ± 118 3576 ± 412

956±383 826 ±47 1500 ±410 982 ± 281 5094 ±286

31 ±12 86 ±24 168 ± 103 212 ± 54 220 ± 136

125±50 87 ±43 341 ± 146 108 ± 26 168 ± 54

100lpg DNP-OVA

Saline 10 pg DNP-OVA 10pgDNP-thymosin

208 ± 37

11,412 ±1256 841 ±109

489 ± 142 632 ± 84 927±208

107 ± 39 3986 ± 142 185±28

145 ± 33 237 ± 129 358±66

625 ± 350 316 ±48 18,562 ± 1225

591 ±117 556 ± 184 8231 ± 1381

80 ± 31 129 ± 26 4862 ±617

50 ± 14 212 ± 43 5325 ±2016

100lpg DNP-thymosin

Saline

10Opg DNP-OVA 10pg DNP-thymosin

* On day 0 groups of +/nu and nu/nu mice were immunized with 100 pg of DNP-OVA or 100 pg of DNP-thymosin in a volume of 0-5 ml. Control groups received saline. t On day 14 secondary immunization consisted of an i.p. injection of the appropriate antigen or saline in a volume of 0'5 ml. t Three days after secondary challenge IgM (D-PFC) response of spleen cells from each mouse was determined using TNPburro RBC and each value represents the mean of means ± s.e. of data obtained from four mice. § Five days after secondary challenge IgG (I-PFC) response of spleen cells from each mouse was determined using TNPburro RBC and each value represents the mean of means ± s.e. of data obtained from four mice.

Slater & White, 1966), thymic hormonal factor (Kook & Trainin, 1974), thymin or thymopoietin (Basch & Goldstein, 1974) and thymic factor (Bach & Dardenne, 1973). Many in vitro and in vivo assays have been described for the testing of the biological activity of these products (reviewed in Friedman, 1975). In this communication, we have characterized the immunogenic and hormonal properties of DNP-thymosin fraction 5 as part of our efforts to elucidate the process of helper T cell maturation. The hapten-linked thymosin is clearly immunogenic in normal and thymus-deficient mice. Both athymic (nu/nu) and TxBm mice mount a significant anti-DNP plaque-forming cell response following primary immunization, and when assayed, both direct (19S) and indirect (7S) responses were achieved. Furthermore, data indicate that the ability of DNP-thymosin to trigger an anti-hapten response was not due to polyclonal stimulation, since immunization with DNP-thymosin does not give rise to nonspecific immune anti-SRBC plaques (Table 2). As reported previously, immunization of nude mice with thymosin leads to a dramatic increase in

response of their lymph node cells to the T-cell mitogen, Con A (Thurman et al., 1975). Such assays were performed in these groups of mice (see Table 3) and it was determined that their lymph node cells responded vigorously to Con A after DNP-thymosin administration. Thus, hapten linkage of thymosin does not destroy its ability to act as a hormone. It was therefore believed that the hapten-linked hormone, DNP-thymosin, was able to exert its influence as a hormone on the development and maturation of precursor T cells to helper T cells and thus influence and induce the formation of antiDNP antibodies by the DNP-reactive B cells, either by direct presentation of DNP to the antigenreactive B cells or via the macrophages. Further evidence of the thymic-dependent nature of this hapten carrier comes from the experiment where use was made of a specific strain of mice, the CBA/N, which has been previously characterized to have an X-linked defect in their ability to respond to soluble thymus-independent antigens (Scher et al., 1975). Experiments were thereby carried out with groups of CBA/N, DBA/2, (CBA/N Y x DBA/2 &)Fj male and female mice immunized with either SRBC (known T-dependent antigen), DNP-

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lys-Ficoll (a known T-independent antigen), and DNP-thymosin. Four days later, spleen cells were individually assayed for their ability to form IgM-PFC against SRBC and TNP-burro RBC. The F1 male mice showed a definite response to SRBC and DNP-thymosin although, as expected, their response was lower than normal mice (Janeway & Barthold, 1975) and, as expected, F1 male mice did not respond to DNP-Ficoll. It is clear from this experiment that the mechanism by which DNPthymosin stimulates an immune response is not dependent on those cells, absent in CBA/N and (CBA/N Y x DBA/2 S)F1 male mice, which are involved in the response to DNP-Ficoll. These data, in addition to our inability to detect any polyclonal activity following immunization indicate that DNP-thymosin is not a thymic-independent antigen, but works probably through the maturation of a small percentage of precursor, helper T cells. Another classical attribute of thymic-dependent antigens is their ability to give rise to memory T cells; most T-independent antigens do not give rise to a secondary response (Basten & Howard, 1973). Therefore, we compared the ability of DNPthymosin and DNP-OVA to prime and induce an anamnestic response in + /nu and nu/nu mice. Both types of mice give a significant secondary response to DNP-thymosin. These data demonstrate the existence of memory T cells after immunization of nu/nu mice with DNP-thymosin and not DNP-OVA, indicating some conversion of precursor T cells to memory T cells by the presentation of DNP on a thymosin carrier molecule. Since the thymosin used consisted of several polypeptides, it is not known from the data in these experiments whether the active polypeptide that is responsible for the conversion of precursor T cells to helper T cells for a thymic-dependent, antibody response to DNP is the same polypeptide that is presented to the clone of antigen-reactive B cells, either directly or via macrophages. Current experiments are being undertaken using purified, single polypeptides to determine the sequence of events that lead to the generation of helper T cells and the anti-hapten-specific response. It is entirely possible that of the several polypeptides known to comprise thymosin, some may act to induce the maturation of precursor cells and others to present determinants to antigenic reactive cells. It is also a possibility that the various polypeptides in thymosin act in concert to bring about the maturation of immunocompetent cells. The purifica-

tion of these single polypeptides and the subsequent biological relationships of each are currently under investigation. ACKNOWLEDGMENTS

This work was supported in part by the Naval Medical Research and Development Command, Work Unit No. MR041.02.01.0034, the National Cancer Institute grants CA 14108 and CA 16964, the John H. Hartford Foundation, Inc., and Hoffman La-Roche, Inc., Nutley, New Jersey. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the 'Guide for the Care and Use of Laboratory Animals', Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 74-23. The authors wish to gratefully thank Mr Timothy E. Brown for his valuable technical assistance and Mrs Joan E. Budd for her excellent editorial assistance. REFEREN CES AHMED A., SCHER I. & SELL K.W. (1977) Studies on nonH-2-linked lymphocyte activating determinants. IV. Ontogeny of the MIs product on murine B cells. Cell.

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Thymic-dependent anti-hapten response in congenitally athymic (nude) mice immunized with DNP-thymosin.

Immunology 1977 33 757 Thymic-dependent anti-hapten response in congenitally athymic (nude) mice inmunized with DNP-thymosin A. AHMED*, A. H. SMITH*,...
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