Is Much Greater in the Bone Marrow Than in the Spleen of Antigenically Unstimulated Adult Animals

THE EFFECT OF THYMIC ACTION ON THE PRECURSORS OF ANTIGEN-SENSITIVE CELLS* BY G. DORIA AND G. AGAROSSI

C. N. E. N.-EURATOM 1MMUNOGENETICS GROUP, LABORATORY OF ANIMAL RADIOBIOLOGY, CENTER FOR NUCLEAR STUDIES, CASACCIA (ROME), ITALY Communicated by Albert H. Coons, July 27, 1967 It is well established that thymectomy in early life impairs the development of the immune response. 1-4 Like replacement of intact thymus, transplantation of dissociated cells from lymph nodes, spleen, or thoracic duct, but not from bone marrow, can prevent the effects of thymectomy." I The immunologic effects of thymectomy performed in adult life are less evident,7-9 but become appreciable under some experimental conditions. Recovery from immunologic paralysis'0 or from sublethal radiation damage of the immunologic potential" is remarkably affected by thymectomy in adult life. In lethally irradiated mice, recovery of the immune system by transplantation of hemopoietic cells may also be conditioned by the host thymus, depending on the tissue origin of the transplanted cells. Unlike spleen cells, bone marrow or fetal liver cells fully require the thymus to establish adoptive immunity in irradiated hosts. 12-14 These observations on adoptive immunity in neonatally thymectomized mice and irradiated mice thymectomized in adult life suggested that in the mouse there are populations of potentially competent cells with different degrees of thymus dependence. In a subsequent study'5 of the agglutinin responses to sheep and rat red blood cells by isogenic bone marrow or spleen cells transplanted in thymectomized and nonthymectomized lethally irradiated mice, the thymus dependence of donor cells was quantitatively assessed and found to be remarkably greater for bone marrow than for spleen cells. The difference in thymus dependence between bone marrow and spleen cells was accounted for by the following hypothesis on the role of the thymus in the process of antibody formation. Potentially competent cells (PC) consist of two cell compartments: antigen-sensitive cells (PC,) and their precursors (PCo). The thymus promotes differentiation of PCo to PC, cells, a process which does not require antigenic stimulation. Antigen triggers off differentiation of PC, cells to antibody-forming cells, as in the model set forth by Makinodan and Albright.'6 The pool size of PCo cells (thymus-dependent) relative to that of PC, cells (thymus-independent) is much greater in the bone marrow than in the spleen of antigenically unstimulated adult animals. This hypothesis was tested in the present work by retransplantation experiments. The results strongly support the hypothesis that the thymus acts on potentially competent cells by promoting the differentiation of PCo to PC, cells. Materials and Methods.-Mice: Animals were male and female C3HeB/FeJ mice obtained periodically from the animal production stock of the Jackson Memorial Laboratory, Bar Harbor, Maine. Upon arrival in our laboratory, animals were kept in quarantine for a month before experimental use. Mice of either sex were caged in groups of five and allowed free access to pellets of standard food and chlorinated water. Thymectomy: Ten- to fourteen-week-old mice were thymectomized or sham-operated as described by Miller.'7 At sacrifice, thymectomized mice were autopsied and those with thymus remnants discarded. Irradiation: Twelve- to sixteen-week-old normal mice and mice surgically treated two weeks 1366 Downloaded by guest on September 27, 2021 VOL. 58, 1967 PATHOLOGY: DORIA AND AGAROSSI 1367

earlier were given a total-body X-ray dose of 800 r. Irradiation conditions were the same as previously described.'5 All mice of repeated radiation-control experiments died within the second week after irradiation. Cell suspensions: Cells were prepared from pools of bone marrow, spleen, or lymph nodes from donors of either sex. (a) Bone marrow: Excised femurs were cut at both ends and flushed through with Tyrode's solution. Marrow plugs were disrupted by aspiration through 22- and 27-gauge needles. (b) Spleen: Spleens were dissected out and teased apart in Tyrode's solution. Cell clumps were dissociated by aspiration through needles as described above. (c) Lymph nodes: Mesenteric lymph nodes were cut in small pieces in Tyrode's solution and then dissociated by aspiration through 18- and 22-gauge needles. After centrifugation at 500 X g for 10 min, the pellet-was resuspended in Tyrode's solution and passed through a 27-gauge needle. Each cell suspension was filtered through a stainless-steel mesh screen. Nucleated cells were counted in a hemoeytometer and volumes adjusted to the desired cell concentrations. Serology: The antigen used was sheep red blood cells (RBC) prepared as previously described" from sheep blood in Alsever's solution purchased from Colorado Serum Co., Denver, Colorado. Sheep RBC at the concentration of 1% (v/v) in phosphate-buffered saline were used for animal immunizations and serum titrations. Mice were bled from the ophthalmic venous plexus or by decapitation. Individual sera were twofold serially diluted and tested for anti-sheep RBC agglutinins.1" The titer was defined as the reciprocal of the highest dilution giving a persistent macroscopic agglutination. Titers were converted to log,, which represent dilution tube numbers suitable for statistical analysis. Experimental design: Normal marrow or spleen cells were cultured in thymectomized and nonthymectomized lethally irradiated mice for a sufficient time to allow repopulation of the hemopoietic tissues by donor cells. Cells from these tissues were retransplanted to and antigenically stimulated in a second series of thymectomized and nonthymectomized lethally irradiated mice to evaluate the thyitus dependence of the immune response of the retransplanted cells. Technical details follow. Bone marrow: One ml of a cell suspension containing 5 X 106 nucleated bone marrow cells from 12- to 16-week-old normal donors was injected intravenously (I.V.) into thymectomized and nonthymectomized lethally irradiated mice (intermediate recipients) within 2 hr after irradiation. The intermediate recipients of both groups were sacrificed a month later. Lymph node, spleen, and bone marrow cell suspensions were prepared, each from a pool of equal tissues (intermediate tissues) harvested from intermediate recipients of either group. Half a ml of each of the six cell suspensions containing 5 X 106 nucleated cells was injected I.V. into thymectomized and nonthymectomized irradiated mice (test recipients) within 2 hr after irradiation. Injection of the lymph node cells was immediately followed by I.V. injection of 0.5 ml of a cell suspension containing 2 X 106 nucleated bone marrow cells from normal donors, in order to ensure long-term survival of these test recipients. Sixty and 66 days after the transplantation of cells from each intermediate tissue, thymectomized and nonthymectomized test recipients were injected intraperi- toneally (I.P.) with 1 ml of 1% sheep RBC and bled 6 days after each antigen injection for serum titration of anti-sheep RBC agglutinins. At the first bleeding, 0.5 ml of blood was withdrawn from the ophthalmic venous plexus. At the second bleeding, blood was collected by decapitation. Spleen: The experimental design was the same as that described above, in all details but two concerning the cell dosage: (1) Intermediate recipients were given 50 X 106 nucleated spleen cells from normal donors. (2) Test recipients were given 20 X 106 nucleated cells from intermediate tissues. Preliminary experiments: (1) Unoperated and sham-thymectomized lethally irradiated mice were given bone marrow or spleen cells from normal donors and sheep RBC 60 and 66 days after the cell transfer. The 6-day primary and secondary agglutinin responses were both the same in the unoperated and sham-thymectomized groups. (2) Nonthymectomized and thymectomized lethally irradiated mice were given 2 X 106 nucleated bone marrow cells from normal donors and sheep RBC 60 and 66 days after the cell transfer. The 6-day mean log, titers and standard errors for the primary and secondary agglutinin responses were 5.3 i 0.7 and 7.5 i 0.1 in the nonthymectomized group, 0.8 i 0.5 and 0.7 i 0.7 in the thymectomized group. (3) In a pilot retransplantation experiment, 5 X 106 (instead of 50 X 106) nucleated spleen cells from normal Downloaded by guest on September 27, 2021 1368 PATHOLOGY: DORIA AND AGAROSSI PROC. N. A. S.

donors were injected into nonthymectomized irradiated intermediate recipients. One month later, 5 X 106 (instead of 20 X 106) nucleated bone marrow cells from the intermediate recipients were injected into 20 thymectomized and 20 nonthymectomized irradiated test recipients. These recipients were given sheep RBC 60 and bled 66 days after the cell transfer. No anti-sheep RBC agglutinins could be detected in the serum of the 4 nonthymectomized and 15 thymectomized test recipients that survived. Results.-Bone marrow: As shown in Table 1, all retransplanted cells were able to respond to sheep RBC when stimulated in thymectomized and nonthymectomized lethally irradiated test recipients. The agglutinin titer in both groups of test recipients was always greater in the secondary than the primary response. Primary and secondary responses were lower in thymectomized than in nonthymectomized test recipients. The difference between mean log2 titers in these recipients was taken as an estimate of the thymus dependence of the immune response by retransplanted cells. The thymus dependence of retransplanted cells from lymph nodes was probably overestimated, since the response of 2 X 106 normal bone marrow cells added to the lymph node cell inoculum might have contributed to the titer in nonthymectomized and thymectomized test recipients unevenly, as expected from the results of preliminary experiment (2). In order to determine whether bone marrow cells grown for a month in the presence or absence of thymus had developed a different degree of thymus dependence, single or pooled values of the thymus dependence of cells from tissues of thymectomized and nonthymectomized intermediate recipients were compared. Differences in thymus dependence and their statistical significance are reported in Table 2. Probability values are referred to the one-tailed t test, in which variances of the titration data were taken into account. If P is set at 0.01, one is allowed to state the following. The pooled thymus dependence of cells from lymph node, spleen, and marrow of thymectomized intermediate recipients was greater than the pooled thymus dependence of cells from the same tissues of nonthymectomized intermediate recipients. The magnitude of the difference in thymus dependence, as found in the secondary response, corresponds to a drop in agglutinin titer of about 300-fold. When single tissues of thymectomized and nonthymectomized intermediate recipients were compared, the difference between the thymus dependences of cells from lymph nodes or spleens was found statistically significant, while the difference between the thymus dependences of cells from bone marrow was below the threshold of significance. Spleen: Table 3 presents the primary and secondary responses to sheep RBC by retransplanted cells of spleen origin. Adoptive immunity in nonthymectomized and thymectomized test recipients has the same general features already described for cells of marrow origin. As in the preceding retransplantation experiment, differences between the thymus dependences of cells from intermediate tissues were similarly computed and analyzed, and the results are illustrated in Table 4. If the significance level is set at P = 0.01, the following conclusions can be drawn. The pooled thymus dependence of cells from lymph node, spleen, and marrow of thymectomized intermediate recipients was greater than the pooled thymus dependence of cells from the same tissues of nonthymectomized intermediate recipients. The magnitude of the difference in thymus dependence, as found in the secondary response, corresponds to a drop in agglutinin titer of about 30-fold. When single tissues of thymectomized and nonthymectomized intermediate recipients were Downloaded by guest on September 27, 2021 VOL. 58, 1967 PATHOLOGY: DORIA AND AGAROSSI 1369

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compared, the difference between the thymus dependences of cells from the spleen was found statistically significant, while the difference between the thymus dependences of cells from lymph node or bone marrow was below the threshold of significance. Discussion.-The interpretation of the results is based on the assumption that anti-sheep RBC agglutinins found in the test recipient sera were produced only by the retransplanted donor cells. This assumption is supported by the following facts. (a) Preliminary experiment no. (3) showed that when cells from insuffi- ciently repopulated animals were transplanted, some test recipients survived but were unable to respond to sheep RBC. This result proves that the host immune system did not recover within 60 days after irradiation. (b) In both bone marrow and spleen retransplantation experiments, different agglutinin titers were found in thymectomized test recipients when cells from different intermediate tissues were transplanted. This observation indicates that antibodies were produced by donor cells. (c) In a previous study'8 the immune system of long-term mouse radiation chimeras was proved to be only of donor type, regardless of the strain combination used and the tissue origin of the transplanted cells. It was found that anti-sheep RBC agglutinins detected in the serum of lethally irradiated mice given allogeneic bone marrow or fetal liver cells were produced only by donor cells, even if the recipients were antigenically stimulated as late as four months after irradiation and cell transplantation. Bone-marrow-derived cells showed a much lower degree of thymus dependence when cultured for a month in presence rather than in absence of the thymus. The difference between the pooled thymus dependences of retransplanted cells is con- sistent with the hypothesis that the thymus acts on potentially competent cells by promoting the differentiation of thymus-dependent (PC0) to thymus-independent (PC,) cells. Likewise, spleen-derived cells developed a different degree of thymus dependence when grown in presence or absence of the thymus. The difference between the pooled thymus dependences of retransplanted cells, however, was less striking than that for cells of marrow origin, as can be expected from the notion that the ratio of PC0 to PC, cells is smaller in the spleen than in the bone marrow."5 Thus, the results of both retransplantation experiments show that the thymus acts on the development of the precursors of antigen-sensitive cells. The present data do not support the hypothesis that the thymus exerts its influence on the response of antigen-sensitive cells when antigenically stimulated.'9 This possibility has been ruled out by Miller et al.6 who found that antigen-sensitive cells are capable of proliferating and differentiating to antibody-forming cells as efficiently in thymectomized hosts as in sham-operated controls. Comparison of the thymus dependences of cells from single intermediate tissues provides information as to the tissue distribution of differentiated PC, cells. Since the thymus dependence of cells from the bone marrow of thymectomized and nonthymectomized intermediate recipients was found to be the same, it can be con- cluded that in presence of the thymus PC, cells neither migrated to nor probably originated in the bone marrow. The difference between the thymus dependences of cells from the spleens of intermediate recipients indicates that PC, cells, derived from PC0 cells in the nonthymectomized intermediate recipients, originated in the spleen or migrated to it from elsewhere. While for these tissues the same conclu- Downloaded by guest on September 27, 2021 1372 PATHOLOGY: DORIA AND AGAROSSI PROC. N. A. S.

sions can be drawn from both retransplantation experiments, the results of retrans- planting cells from lymph nodes must be considered separately. In the bone marrow retransplantation experiment, the different thymus dependence of cells from lymph nodes of thymectomized and nonthymectomized recipients clearly shows that thymus-induced PC1 cells were present in lymph nodes, a finding com- patible with cell migration or local differentiation. In the spleen retransplantation experiment, no difference was found between the thymus dependence of cells from lymph nodes of thymectomized and nonthymectomized intermediate recipients. The large inoculum of spleen cells (originally rich in PC1 cells), which was required to ensure survival of irradiated mice, might have saturated the PC1 compartment of the intermediate lymph nodes, thus preventing local differentiation or migration of new PC1 cells. The present work, while showing that the thymus induces differentiation of PCo cells of marrow and spleen origin, does not indicate whether this process occurs in the thymus or in other lymphoid tissues. The site of differentiation of PCo cells is now being investigated. Summary.-The thymus acts on potentially competent cells by triggering the differentiation of the precursors of antigen-sensitive cells. Thymus-induced antigen-sensitive cells were found distributed in the spleen and lymph nodes, but not in the bone marrow.

We are grateful to Mr. Sergio Di Pietro for excellent technical assistance. * Work supported by C.N.E.N.-Euratom Association contract. Publication no. 330 of the Euratom Biology Division. 1 Miller, J. F. A. P., Lancet, II, 748 (1961). 2 Archer, 0., and J. C. Pierce, Federation Proc., 20, 26 (1961). 3 Arnason, B. G., B. D. Jankovic, and B. H. Waksman, J. Exptl. Med., 116, 177 (1962). 4 Dalmasso, A. P., C. Martinez, and R. A. Good, Proc. Soc. Exptl. Biol. Med., 110, 205 (1962). 6 Miller, J. F. A. P., Science, 144, 1544 (1964). 6i Miller, J. F. A. P., G. F. Mitchell, N. S. Weiss, Nature, 214, 992 (1967). 7Taylor, R. B., Nature, 208, 1334 (1965). 8 Metcalf, D., Nature, 208, 1336 (1965). 9 Miller, J. F. A. P., Nature, 208, 1337 (1965). 10Claman, H. N., and D. W. Talmage, Science, 141, 1193 (1963). 11 Miller, J. F. A. P., Nature, 195, 1318 (1962). 12Miller, J. F. A. P., S. M. A. Doak, and A. M. Cross, Proc. Soc. Exptl. Biol. Med., 112, 785 (1963). 13 Duplan, J. F., Comp. Rend., 256, 3366 (1963). 14 Cross, A. M., E. Leuchars, and J. F. A. P. Miller, J. Exptl. Med., 119, 837 (1964). 16 Doria, G., and G. Agarossi, Tranzplantation, submitted. '6 Makinodan, T., and J. F. Albright, in Immunopathology, Third International Symposium, La Jolla, 1963 (Basel, Switzerland: Schwabe and Co. Publ., 1963), p. 99. 17Miller, J. F. A. P., Brit. J. Cancer, 14, 93 (1960). 18 Doria, G., J. W. Goodman, N. Gengozian, and C. C. Congdon, J. Immunol., 88, 20 (1962). 19 Metcalf, D., and M. Brumby, J. Cell. Phyeiol., 67 (Sup. 1), 149 (1966). Downloaded by guest on September 27, 2021