CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : I. HEMOLYSIN-FORMING CELLS IN NEONATALLY THYMECTOMIZED MICE RECONSTITUTED WITH THYMUS OR THORACIC DUCT LYMPHOCYTES

An injection of viable thymus or thoracic duct lymphocytes was absolutely essential to enable a normal or near-normal 19S liemolysin-forming cell response in the spleens of neonatally thymectomized mice challenged with sheep erythrocytes. Syngeneic thymus lymphocytes were as effective as thoracic duct lymphocytes in this system and allogeneic or semiallogeneic cells could also reconstitute their hosts. No significant elevation of the response was achieved by giving either bone marrow cells, irradiated thymus or thoracic duct cells, thymus extracts or yeast. Spleen cells from reconstituted mice were exposed to anti-H2 sera directed against either the donor of the thymus or thoracic duct cells, or against the neonatally thymectomized host. Only isoantisera directed against the host could significantly reduce the number of hemolysin-forming cells present in the spleen cell suspensions. It is concluded that these antibody-forming cells are derived, not from the inoculated thymus or thoracic duct lymphocytes, but from the host. Thoracic duct cells from donors specifically immunologically tolerant of sheep erythrocytes had a markedly reduced restorative capacity in neonatally thymectomized recipients challenged with sheep erythrocytes. These results have suggested that there are cell types, in thymus or thoracic duct lymph, with capacities to react specifically with antigen and to induce the differentiation, to antibody-forming cells, of hemolysin-forming cell precursors derived from a separate cell line present in the neonatally thymectomized hosts.     

ANTIGEN-SPECIFIC SYNERGISM IN THE IMMUNE RESPONSE OF IRRADIATED MICE GIVEN MARROW CELLS AND PERITONEAL CAVITY CELLS OR EXTRACTS

A synergistic immune response to foreign erythrocytes may be induced in heavily irradiated mice injected with a mixture of isologous cells obtained from marrow and from the peritoneal cavity. Under appropriate conditions, homologous or heterologous peritoneal cavity cells, heat-killed cells, or cellfree extracts made from such cells are also effective. The activity of the peritoneal cavity cells or extracts is antigen-specific, in the sense that cells or extracts obtained from animals previously immunized with the test antigen produce much stronger synergistic effects than do cells from animals immunized with some other antigen; however, the peritoneal cavity cells or extracts are not immunogenic when tested in primed animals. The marrow cells, demonstrated to contain precursors of the antibody-forming cells produced during this synergistic immune response, also show a form of antigen-specificity.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : III. CHROMOSOMAL MARKER ANALYSIS OF SINGLE ANTIBODY-FORMING CELLS IN RECONSTITUTED, IRRADIATED, OR THYMECTOMIZED MICE

Two new methods are described for making chromosomal spreads of single antibody-forming cells. The first depends on the controlled rupture of cells in small microdroplets through the use of a mild detergent and application of a mechanical stress on the cell. The second is a microadaptation of the conventional Ford technique. Both methods have a success rate of over 50%, though the quality of chromosomal spreads obtained is generally not as good as with conventional methods. These techniques have been applied to an analysis of cell to cell interaction in adoptive immune responses, using the full syngeneic transfer system provided by the use of CBA and CBA/T6T6 donor-recipient combinations. When neonatally thymectomized mice were restored to adequate immune responsiveness to sheep erythrocytes by injections of either thymus cells or thoracic duct lymphocytes, it was shown that all the actual dividing antibody-forming cells were not of donor but of host origin. When lethally irradiated mice were injected with chromosomally marked but syngeneic mixtures of thymus and bone marrow cells, a rather feeble adoptive immune response ensued; all the antibody-forming cells identified were of bone marrow origin. When mixtures of bone marrow cells and thoracic duct lymphocytes were used, immune restoration was much more effective, and over three-quarters of the antibody-forming mitotic figures carried the bone marrow donor chromosomal marker. The results were deemed to be consistent with the conclusions derived in the previous paper of this series, namely that thymus contains some, but a small number only of antigen-reactive cells (ARC), bone marrow contains antibody-forming cell precursors (AFCP) but no ARC, and thoracic duct lymph contains both ARC and AFCP with a probable predominance of the former. A vigorous immune response to sheep erythrocytes probably requires a collaboration between the two cell lineages, involving proliferation first of the ARC and then of the AFCP. The results stressed that the use of large numbers of pure thoracic duct lymphocytes in adoptive transfer work could lead to good adoptive immune responses, but that such results should not be construed as evidence against cell collaboration hypotheses. Some possible further uses of single cell chromosome techniques were briefly discussed.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : IV. THE RESPONSE OF NORMAL AND IMMUNE RABBIT BONE MARROW AND LYMPHOID TISSUE LYMPHOCYTES TO ANTIGENS IN VITRO

Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VI. THE IMMUNE RESPONSE TO RED BLOOD CELLS IN IRRADIATED RABBITS AFTER ADMINISTRATION OF NORMAL, PRIMED, OR IMMUNE ALLOGENEIC RABBIT BONE MARROW CELLS

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.     

PROLIFERATION OF DONOR MARROW AND THYMUS CELLS IN THE MYELOID AND LYMPHOID ORGANS OF IRRADIATED SYNGENEIC HOST MICE

CBA/HT₆T₆ bone marrow cells (1 x 10⁷) or CBA/H bone marrow cells (1 x 10⁷) plus CBA/HT₆T₆ thymus cells (5 x 10⁷) were injected intravenously into lethally (800 R) irradiated CBA/H mice. Chromosome analyses of dividing cells in the host lymphoid and myeloid organs were performed at intervals after irradiation. Donor marrow cells settled and proliferated in the host bone marrow, spleen, and lymph nodes soon after injection, but donor marrow cells did not proliferate in the host thymus until day 10; then host-type cells were quickly replaced by donor-type cells in the thymus by day 20. On the other hand, donor thymus cells settled and proliferated in the host thymus and lymph nodes soon after injection but they gradually disappeared from these organs. On day 20, a few donor-type dividing cells (of thymus origin) were found in the host lymphoid and myeloid organs.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : V. TARGET CELLS FOR TOLERANCE INDUCTION

Collaboration between thymus-derived lymphocytes, and nonthymus-derived antibody-forming cell precursors occurs during the immune response of mice to sheep erythrocytes (SRBC). The aim of the experiments reported here was to attempt to induce tolerance in each of the two cell populations to determine which cell type dictates the specificity of the response. Adult mice were rendered specifically tolerant to SRBC by treatment with one large dose of SRBC followed by cyclophosphamide. Attempts to restore to normal their anti-SRBC response by injecting lymphoid cells from various sources were unsuccessful. A slight increase in the response was, however, obtained in recipients of thymus or thoracic duct lymphocytes and a more substantial increase in recipients of spleen cells or of a mixture of thymus or thoracic duct cells and normal marrow or spleen cells from thymectomized donors. Thymus cells from tolerant mice were as effective as thymus cells from normal or cyclophosphamide-treated controls in enabling neonatally thymectomized recipients to respond to SRBC and in collaborating with normal marrow cells to allow a response to SRBC in irradiated mice. Tolerance was thus not achieved at the level of thelymphocyte population within the thymus, perhaps because of insufficient penetration of the thymus by the antigens concerned. By contrast, thoracic duct lymphocytes from tolerant mice failed to restore to normal the response of neonatally thymectomized recipients to SRBC. Tolerance is thus a property that can be linked specifically to thymus-derived cells as they exist in the mobile pool of recirculating lymphocytes outside the thymus. Thymus-derived cells are thus considered capable of recognizing and specifically reacting with antigenic determinants. Marrow cells from tolerant mice were as effective as marrow cells from cyclophosphamide-treated or normal controls in collaborating with normal thymus cells to allow a response to SRBC in irradiated recipients. When a mixture of thymus or thoracic duct cells and lymph node cells was given to irradiated mice, the response to SRBC was essentially the same whether the lymph node cells were derived from tolerant donors or from thymectomized irradiated, marrow-protected donors. Attempts to induce tolerance to SRBC in adult thymectomized, irradiated mice 3–4 wk after marrow protection, by treatment with SRBC and cyclophosphamide, were unsuccessful: after injection of thoracic duct cells, a vigorous response to SRBC occurred. The magnitude of the response was the same whether or not thymus cells had been given prior to the tolerization regime. The various experimental designs have thus failed to demonstrate specific tolerance in the nonthymus-derived lymphocyte population. Several alternative possibilities were discussed. Perhaps such a population does not contain cells capable of dictating the specificity of the response. This was considered unlikely. Alternatively, tolerance may have been achieved but soon masked by a rapid, thymus-independent, differentiation of marrow-derived lymphoid stem cells. On the other hand, tolerance may not have occurred simply because the induction of tolerance, like the induction of antibody formation, requires the collaboration of thymus-derived cells. Finally, tolerance in the nonthymus-derived cell population may never be achieved because the SRBC-cyclophosphamide regime specifically eliminates thymus-derived cells leaving the antibody-forming cell precursors intact but unable to react with antigen as there are no thymus-derived cells with which to interact.     

THE ROLE OF THYMUS AND BONE MARROW CELLS IN DELAYED HYPERSENSITIVITY

Adoptive transfer experiments were performed to define the immunological role of thymus and bone marrow cells in the induction of delayed hypersensitivity (DH). The results indicated the following, (a) Bone marrow from immune donors contained cells capable of being stimulated by antigen to initiate the expression of DH. (b) Bone marrow from nonimmune or tolerant donors contained cells that were needed to complete the expression of DH after the infusion of immune lymph node cells. (c) Normal bone marrow and thymus cells cooperated in the irradiated recipient to induce the most vigorous skin reactions to specific antigen; these reactions were seen only when the recipients were stimulated by antigen. Either cell type alone was ineffective. (d) In the presence of tolerant bone marrow cells, thymus cells from immune donors gave a more vigorous response than did thymus cells from normal or tolerant donors. (e) There was suggestive evidence that thymus cells were the source of trigger elements that initiated DH. (f) Antigen in the irradiated recipient was necessary to induce DH after infusion of bone marrow cells alone, or bone marrow and thymus cells together.     

THE THYMUS AND RECOVERY OF THE SHEEP ERYTHROCYTE RESPONSE IN IRRADIATED MICE

The role of the thymus in the recovery of the sheep erythrocyte response after lethal irradiation has been studied in adult CBA mice with the hemolytic plaque technique of Jerne. This immunological parameter is markedly thymus-dependent. 10 wk after irradiation and after antigenic challenge the thymectomized animal has only one-twentieth to one-fortieth the number of plaque-forming cells as does the irradiated animal with intact thymus. The thymus continues to function into the 7th and 8th month of life in this strain. Unlike the drug-tolerant animal, the incompetent irradiated thymectomized mouse retains base line plaques (plaques without antigenic stimulation). Thymectomy 18 days after irradiation is as effective as prior thymectomy in preventing recovery of the sheep cell response. Thymectomized animals receiving grafts of isogenic neonatal thymus (placed beneath the kidney capsule) 1 day, 1 wk, or 2 wk after irradiation are somewhat more responsive at 10 wk than intact animals. Grafts in place for 1 or 2 wk after irradiation and then removed result in one-fifth the recovery of grafts in place the entire time, while only slight restoration is obtained from grafts in place for the final 3 wk of the experiment. The results indicate that the thymus is not required for the 18 days after irradiation, that a period of at least 3 wk residence is required for complete restoration, and that the thymus itself is somewhat radiation-sensitive. Allogeneic thymus grafts failed to restore the hemolysin response of irradiated thymectomized animals.     

ALLOGENEIC THYMUS GRAFTS AND THE RESTORATION OF IMMUNE FUNCTION IN IRRADIATED THYMECTOMIZED MICE

Irradiated and thymectomized CBA mice are markedly depressed in several immunological parameters (skin homograft rejection, graft-vs.-host activity and hemolytic plaque-forming cells of the spleen, hemolysin and hemagglutinin formation, and peripheral lymphocyte counts). In the present experiments the ability of homografts of neonatal thymus placed beneath the kidney capsule to restore immunological capacity of such animals was studied. Thymus homografts which share the same H-2 locus with the CBA mouse were permanently tolerated and immunological restoration was complete. Skin from the thymus donor was specifically retained, but third party skin with even minor (non-H-2) incompatibility was normally rejected and hemolytic plaque-forming cells of the spleen were restored. Thymus homografts which differ at the H-2 locus were promptly rejected and led to accelerated rejection of skin subsequently grafted from the thymus donor. With such H-2 incompatible thymus grafts, third party skin with minor histo-incompatibility was retained while there was slight to moderate restoration of rejection of skin with major (H-2) incompatibility. Graft-vs.-host activity was restored, but there was no return of plaque-forming spleen cells, hemolysins, hemagglutinins, or peripheral lymphocyte counts. In view of the cross-reactivity at the H-2 locus in CBA mice between thymus and third party skin donors, it was felt that restoration of skin rejection and graft-vs.-host activity could be adequately explained on the basis of immunization by the thymus graft and did not require the postulation of true immune restoration or a thymus hormone.     

EXPERIMENTAL STUDIES UPON LYMPHOCYTES : II. THE ACTION OF IMMUNE SERA UPON LYMPHOCYTES AND SMALL THYMUS CELLS.

The work of previous investigators gives the impression that it is easy to produce sera which both in vitro and upon injection are leukotoxic. At the same time the specificity of these leukotoxic sera for the particular type of cell used as antigen, and even for leukocytes in general, has been doubtful. The methods used have made certain possible factors of error unavoidable. Even careful washing of an organ or suspension cannot render it wholly blood-free, so that it is not surprising that the sera should be moderately hemolytic and hemagglutinative. Pearce has shown that the injection of very small amounts of blood is sufficient to evoke the production of immune hemolysins. When such sera are injected the lesions, as Pearce states, may be due in part to the production of hemagglutinative thrombi, although this hardly seems to apply to the changes in lymphoid tissue described by Flexner. On the other hand, the lymphotoxic effect of hemolytic sera may be due to the lymphocytes injected with the red cells. Our own experiments indicate that the lymphotoxic and agglutinative factors are to a considerable degree distinct from the hemolytic and hemagglutinative ones, since they can be separated from one another by absorption. Further evidence is presented that the small thymus cells are biologically related to, if not identical with the lymphocytes derived from lymph glands.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : VI. CONTRIBUTION OF THYMUS-DERIVED CELLS AND ANTIBODY-FORMING CELL PRECURSORS TO IMMUNOLOGICAL MEMORY

Collaboration between thymus-derived lymphocytes and nonthymus-derived antibody-forming cell precursors occurs in the primary antibody response of mice to heterologous erythrocytes and serum proteins. The purpose of the experiments reported here was to determine whether collaboration took place in an adoptive secondary antibody response. A chimeric population of lymphocytes was produced by reconstituting neonatally thymectomized CBA mice soon after birth with (CBA x C57BL)F₁ thymus lymphocytes. These mice could be effectively primed to fowl immunoglobulin G (FγG) and their thoracic duct lymphocytes adoptively transferred memory responses to irradiated mice. The activity of these cells was impaired markedly by preincubation with CBA anti-C57BL serum and to a lesser extent by anti-θ-serum. Reversal of this deficiency was obtained by adding T cells in the form of thoracic duct cells from normal CBA mice. Cells from FγG-primed mice were at least 10 times as effective as cells from normal mice or from CBA mice primed to horse erythrocytes. These results were considered to support the concept that memory resides in the T cell population and that collaboration between T and B cells is necessary for an optimal secondary antibody response. Poor antibody responses were obtained in irradiated mice given mixtures of thoracic duct cells from primed mice and of B cells from unprimed mice (in the form of spleen or thoracic duct cells from thymectomized donors). In contrast to the situation with T cells, the deficiency in the B cell population could not be reversed by adding B cells from unprimed mice. It was considered that memory resides in B cells as well as in T cells and that priming probably entails a change in the B cell population which is fundamentally different from that produced in the T cell population.     

THE IMMUNE RESPONSE TO FOREIGN RED BLOOD CELLS AND THE PARTICIPATION OF SHORT-LIVED LYMPHOCYTES

The sequence of morphological changes in the rat spleen following SRBC injection associated with hemolysin production has been correlated with estimates of proliferative activity by splenic lymphatic tissue. Formation of new, reactive germinal centers containing macrophages which engulf nuclear debris is a prominent feature of the response. This is prevented by pretreatment of the animal with cortisol. Indirect evidence is provided that short-lived lymphocytes produced in germinal centers may be a necessary component in the induction of other cells to proliferate and differentiate into hemolysin-producing cells. The reasons are discussed for considering short-lived lymphocytes, such as those produced in the thymus, bone marrow, and germinal centers, as differing from long-lived lymphocytes capable of antibody synthesis.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : VIII. RADIOSENSITIVITY OF THYMUS-DERIVED LYMPHOCYTES

The helper function of carrier-primed T cells was found to be radiosensitive in vivo. The results could not be attributed to interference with the spleen-seeking properties of the irradiated cells. It is suggested that T cell division is essential for the induction of 7S antibody responses in vivo.     

REGULATION OF THE IMMUNE RESPONSE : III. KINETIC DIFFERENCES BETWEEN THYMUS- AND BONE MARROW-DERIVED LYMPHOCYTES IN THE PROLIFERATIVE RESPONSE TO HETEROLOGOUS ERYTHROCYTES

The kinetics of the in vivo response to SRBC was studied in mouse spleen at both the B cell and T cell levels. The B cell response was assayed by following the appearance of antibody-secreting cells in the spleen using the hemolytic plaque assay. The T cell response was monitored by following the increase in or "priming" of helper activity in the spleen using a quantitative in vitro assay. The role of cellular proliferation in both responses was established with the inhibitor of mitosis, vinblastine. The results show that, although the development of T cell activity precedes that of anti-SRBC PFC by as much as 1 day, T cells lag at least 1 day behind B cells in the onset of cellular proliferation. The evidence suggests either that the helper T cell which proliferates in response to SRBC does so after helping in the initiation of the primary B cell response or that the proliferative T cell response and the initiation of the primary B cell response involve two different subpopulations of T cells.     

INDUCTION OF A HEMOLYSIN RESPONSE IN VITRO : INTERACTION OF CELLS OF BONE MARROW ORIGIN AND THYMIC ORIGIN

The immune response to foreign erythrocytes was studied in vitro. Two subpopulations of cells were prepared. One was a population of bone marrow-derived spleen cells, taken from thymectomized, irradiated, and bone marrow-reconstituted mice; there was evidence that most of the precursors of the PFC had been present in this cell population, but few PFC developed in cultures of these cells alone in the presence of immunogenic erythrocytes. Another cell suspension was made from spleens of mice which had been irradiated and injected with thymus cells and erythrocytes; these cells were called educated T cells. The two cell suspensions together allow the formation of PFC in the presence of the erythrocytes which were used to educate the T cells, but not in the presence of noncross-reacting erythrocytes. If bone marrow-derived cells and T cells were kept in culture together with two different species of erythrocytes, and if one of the erythrocytes had been used to educate the T cells, then PFC against each of the erythrocytes could be detected.     

REGULATION OF THE IMMUNE RESPONSE : II. QUALITATIVE AND QUANTITATIVE DIFFERENCES BETWEEN THYMUS- AND BONE MARROW-DERIVED LYMPHOCYTES IN THE RECOGNITION OF ANTIGEN

The specificity of antigen recognition by thymus-derived helper cells (T cells) and antibody was examined in mice, heterologous erythrocyte antigens from sheep (SRBC), goat (GRBC), burro (BRBC), chicken (CRBC), and toad (TRBC) being used. Antibody specificity was tested by a number of functional assays: hemagglutination, hemolysis, and immune suppression. The specificity of T cells was determined by titrating their ability to help the in vitro antitrinitrophenol (TNP) responses of mouse spleen cultures immunized with the hapten coupled to the various test erythrocytes as carrier. Anti-SRBC antibody cross-reacted with GRBC, but not with BRBC, CRBC, or TRBC. In contrast, SRBC-primed helper T cells cross-reacted with both GRBC and BRBC, but not with CRBC or TRBC, indicating a difference in the specificity of antigen recognition between the cellular and the humoral immune responses.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VII. THE DEMONSTRATION, USING ALLOTYPIC MARKERS, OF ANTIBODY FORMATION BY IRRADIATION-RESISTANT CELLS OF IRRADIATED RABBITS INJECTED WITH NORMAL ALLOGENEIC BONE MARROW CELLS AND SHEEP ERYTHROCYTES

Bone marrow cells obtained from rabbits of one allotype were injected into irradiated rabbits of a different allotype. The recipients were also injected with sheep red blood cells, and their spleen cells were tested for plaque-forming capacity 7 days later. Spleen cells of all recipients gave large numbers of plaques as did spleen cells incubated with antiserum, directed toward donor allotype. However, incubation of the recipient spleen cells with antiserum directed toward recipient allotype completely suppressed plaque formation. These results demonstrate that antibody-formation in irradiated recipients of transferred lymphoid cells is a property of the recipient animal and that the antibody-forming cell is relatively irradiation-resistant. It was also demonstrated that only viable normal bone marrow cells are capable of transferring antibody-forming capacity to irradiated recipient rabbits. Neither sonicates nor heat-killed preparations of normal rabbit bone marrow cells possessed this capacity.     

THYMUS INDEPENDENCE OF A COLLAGEN-LIKE SYNTHETIC POLYPEPTIDE AND OF COLLAGEN, AND THE NEED FOR THYMUS AND BONE MARROW-CELL COOPERATION IN THE IMMUNE RESPONSE TO GELATIN

Several inbred mouse strains were screened for their ability to respond to the ordered periodic collagen-like polymer (Pro-Gly-Pro)_n, to the random copolymer (Pro⁶⁶, Gly³⁴)_n, to the protein conjugate Pro-Gly-Pro-ovalbumin, to rat tail tendon collagen, rat tail tendon gelatin, and to Ascaris cuticle collagen. Differences were obtained in the magnitude of the antibody titers towards the above immunogens among the strains tested. The level of the response to the ordered polymer (Pro-Gly-Pro)_n was not similar to that towards the random (Pro⁶⁶, Gly³⁴)_n, confirming differences in the antigenic determinants of the two immunogens. The role of the thymus in the immune response to (Pro-Gly-Pro)_n and (Pro⁶⁶, Gly³⁴)_n as well as to two collagens and gelatin, was studied in order to find out a possible correlation with the structural features of the immunogens. Heavily irradiated recipients were injected with syngeneic thymocytes, marrow cells, or a mixture of both cell populations and were immunized with the above-mentioned antigens. An efficient immune response to the ordered collagen-like (Pro-Gly-Pro)_n was obtained in the absence of transferred thymocytes. The thymus independence of (Pro-Gly-Pro)_n was confirmed when thymectomized irradiated mice were used as recipients. In contrast with these results, cooperation between thymus and marrow cells was necessary in order to elicit an immune response to (Pro⁵⁶, Gly³⁴)_n. Similarly, the immune response to the triple helical collagen was found to be independent of the thymus, whereas for an effective response to its denatured product, gelatin, thymus cells were required. These findings indicate that a unique three-dimensional structure of immunogens possessing repeating antigenic determinants plays an important role in determining the need for cell to cell interaction in order to elicit an antibody response.     

IMMUNOGLOBULINS ON THE SURFACE OF LYMPHOCYTES : II. THE BONE MARROW AS THE MAIN SOURCE OF LYMPHOCYTES WITH DETECTABLE SURFACE-BOUND IMMUNOGLOBULIN

Immunofluorescent studies using live cells from antibody-forming organs and anti-immunoglobulin antibodies demonstrate two populations of small lymphocytes which are differentiated by the presence or absence of Ig on their surface membranes. Most of the lymphocytes with detectable surface Ig appear to derive from cells of the bone marrow, while most of the Ig-negative lymphocytes derive from the thymus. Thus, adult mice thymectomized, lethally irradiated, and transplanted with bone marrow cells showed a normal number of lymphocytes with surface Ig but were depleted of the Ig-negative lymphocytes. Injection of thymocytes into these mice did not result in an increase in the number of lymphocytes with surface Ig in spleen and lymph nodes. Most of the injected thymocytes could be identified by means of histocompatibility markers. Also, the spleen and lymph nodes of neonatally thymectomized mice contained lymphocytes with surface Ig but were depleted of the Ig-negative lymphocytes. Attempts were made to identify light chains on thymocytes by a sensitive radioimmunoassay. In some experiments no light chains were detected and in others a small amount, i.e. no more than 2.6% of the amount present on spleen lymphocytes, could be detected. Whether these low figures are significant or represent a small amount of serum contamination is not clear as yet.