The Journal of Experimental Medicine

Shearer, G. M.; Mozes, Edna; Sela, Michael

doi: 10.1084/jem.135.5.1009pmid: 4112259

Genetic regulation of immunological responsiveness was studied at the cellular level by comparing the limiting dilutions of immunocompetent cells from spleen, thymus, and bone marrow of high and low responders as a function of the poly- L -prolyl and poly- DL -alanyl side chains of two synthetic polypeptide immunogens. The spleens of immunized and unimmunized high responder DBA/1 mice were found to contain respectively, 18- and 7-fold more limiting precursor cells specific for (Phe, G)-A--L than the spleens of SJL low responder donors. These results, using a synthetic polypeptide built on multichain poly- DL -alanine, confirm the findings reported for polypeptides built on multichain poly- L -proline (1, 2), that there is a direct correlation between immune response potential and the relative number of immunocompetent precursors stimulated. Cell cooperation between thymocytes and bone marrow cells was demonstrated for both (T, G)-Pro--L and (Phe, G)-A--L. Limiting dilutions of thymus and bone marrow cells in the presence of an excess amount of the complementary cell type indicated an eightfold lower number of detected (T, G)-Pro--L-specific precursors in DBA/1 (low responder) marrow when compared with SJL (high responder) marrow. No differences were observed in the frequency of relevant high and low responder thymocytes for the (T, G)-Pro--L immunogen. These results are similar to those reported for the (Phe, G)-Pro--L (3). In contrast to the cellular studies reported for the Pro--L series of immunogens, the marrow and thymus cell dilution experiments for (Phe, G)-A--L revealed genetically associated differences in both the marrow and thymus populations of immunocytes from high (DBA/1) and low (SJL) responders. In addition to a fivefold difference in limiting marrow cell precursors (similar to that seen in the Pro--L studies), a striking difference was observed between the helper cell activity of high responder DBA/1 and low responder SJL thymocytes. This difference was indicated by the observation that low responder thymocyte dilutions followed the predictions of the Poisson model, whereas dilutions of high responder thymocytes did not conform to Poisson statistics. Transfers of allogeneic thymus and marrow cell mixtures from DBA/1 and SJL donors confirmed the syngeneic dilution studies showing that the genetic defect of immune responsiveness to (Phe, G)-A--L is expressed at both the thymus and marrow immunocompetent cell level. The parameters presently known for genetic control of immune responses specific for (Phe, G) ( Ir-1 gene) and for Pro--L ( Ir-3 gene) have been compared. The Ir-1 and Ir-3 genes are not only distinct by genetic linkage tests (to H-2 ) (5, 6, 9), but they are also seen to be different by cellular studies. Furthermore, expression of low responsiveness within a given cell population was shown to depend on the chemical structure of the whole immunogenic macromolecule. Footnotes Submitted: 29 November 1971

Miller, Harold C.; Cudkowicz, Gustavo

doi: 10.1084/jem.135.5.1028pmid: 4553850

Individual immunocompetent precursor cells of (C57BL/10 x C3H)F 1 mouse marrow generate, on transplantation, three to five times more antibody-forming cells localized in recipient spleens during secondary than during primary immune responses. The increased burst size is immunologically specific since antigens of horse and chicken erythrocytes and of Salmonella typhimurium do not cause this effect in marrow cells responsive to sheep red blood cells. Both sensitized and nonsensitized precursors require the helper function of thymus-derived cells and antigen for the final steps of differentiation and maturation. The burst size of primed precursor cells is the same after cooperative interactions with virgin or educated helper cells of thymic origin. The greater potential of these marrow precursors may be attributable to self-replication and migration before differentiation into antibody-forming descendants. In fact, the progeny cells of primed precursor units are distributed among a multiplicity of foci, whereas those of nonimmune precursors are clustered into one focus. The described properties of specifically primed marrow precursors are those underlying immunologic memory. It remains to be established whether memory cells are induced or selected by antigens and whether the thymus plays a role in this process. Footnotes Submitted: 26 December 1971

Rosenstreich, David L.; Shevach, Ethan; Green, Ira; Rosenthal, Alan S.

doi: 10.1084/jem.135.5.1037pmid: 4623313

In this study, the frequency of uropod formation and the type of lymphocyte bearing the uropod was investigated in various guinea pig lymphocyte populations. Without prior in vitro stimulation, almost 40% of peritoneal exudate lymphocytes (PELS) form uropods, while thymocytes and lymph node cells form far fewer. Subsequent stimulation in vitro with purified protein derivative demonstrated that there is an association between antigen reactivity and frequency of uropod formation in these populations. The ultrastructure of these uropods is identical to that described for human lymphocytes stimulated with phytohemagglutinin. In the populations studied, all the lymphocytes forming uropods lack easily detectable surface membrane immunoglobulin and are therefore most likely thymus-derived or T lymphocytes. Footnotes Submitted: 29 December 1971

Feldmann, Marc

doi: 10.1084/jem.135.5.1049pmid: 4112260

The requirement for macrophages in thymus-dependent antibody responses was studied in vitro. Three different macrophage-deficient cell populations were studied: spleen cells passed through a glass bead column at 37°C, spleen cells cultured with specific antimacrophage serum, and thoracic duct lymphocytes. These cell populations from mice primed to dinitrophenylated (DNP) fowl gamma globulin were unable to respond to the homologous conjugate in vitro. DNP-reactive B cells were present in normal proportions, since all three macrophage-depleted populations responded normally to macrophage-independent and thymus-independent DNP flagella. Carrier-reactive T cells were present, as the helper capacity of carrier-primed spleen cells was the same as carrier-primed lymphocytes, and thoracic duct lymphocytes are a well-established source of helper cells. The inhibition of the cooperative response was thus due to removal of macrophages, and this was proven by restoration of thymus-dependent anti-DNP responses by small numbers of anti-θ-treated peritoneal exudate cells. These results suggest that macrophages are essential in cell collaboration, While their exact function in cell collaboration is not yet known, the above observation suggests that the mechanism of T-B collaboration involves the surface of macrophages. Footnotes Submitted: 26 December 1971

Tigelaar, Robert E.; Asofsky, Richard

doi: 10.1084/jem.135.5.1059pmid: 4401814

A mortality assay was used to quantitate graft- versus -host (GVH) reactions in sublethally irradiated (400 R) neonatal (C57BL/6 x BALB/c)F 1 recipients of BALB/c lymphoid cells from various tissues. The probit of the 35 day cumulative per cent of mortality was a linear function of the logarithm of the cell inoculum for any tissue; reactivities of different tissues fell on a series of parallel lines. Peripheral blood leukocytes (PBL), the most active cells, were about 30 times as active as thymocytes, the least active cells studied; femoral lymph node cells and spleen cells were about 23 and 8 times as reactive as thymocytes, respectively. The average survival time of recipients of thymocytes who eventually died was nearly a week longer than that of recipients of comparably lethal numbers of PBL, lymph node, or spleen cells. Mixtures of PBL and thymocytes gave levels of 35 day mortality significantly greater than those expected if the reactivities of the mixture had been merely the sum of the reactivities of the components measured separately, thereby confirming in any assay independent of host splenomegaly the synergistic interaction of thymocytes and PBL in the GVH reaction. Both populations of cells in the mixture had to be allogeneic to the host in order to observe this synergy. The kinetics of cumulative mortality observed for mixtures of PBL and thymocytes were indistinguishable from those seen with thymocytes alone, indicating activation of the latter cell type. Finally, comparison of the relative abilities of different cell populations to cause splenomegaly on the one hand and lethal runting on the other has raised the possibility that expression of different effector functions of cell-mediated immune reactions may in fact be initiated by distinct cells. Footnotes Submitted: 2 January 1972

Biozzi, G.; Stiffel, C.; Mouton, D.; Bouthillier, Y.; Decreusefond, C.

doi: 10.1084/jem.135.5.1071pmid: 4553851

Two lines of mice have been separated by selective breeding for the character "agglutinin production to heterologous erythrocytes." Around the 18th generation of selection the two lines could be considered as homozygous for the character investigated. This trait is under the control of a group of additive genes. The interline difference in the production of anti-SE agglutinins was verified for the range of antigen doses from subimmunogenic to maximal. After intravenous immunization with an optimal dose of SE, the duration of the exponential rise in serum antibody was 4–5 days in both lines. At this time most of the interline difference in responsiveness is already expressed. A cytodynamic study carried out in terms of plaque-forming cells (PFC) and rosette-forming cells (RFC) in the spleen during the exponential phase showed that the principal interline difference is found in the doubling time of cells engaged in the immune response. More precise cytodynamic analysis made in terms of RFC showed that the doubling time of RFC is 9 hr in high responder and 16 hr in low responder mice. The duration of the exponential rise and the number of target cells stimulated by antigen is the same in both lines. The interline difference at the end of the exponential rise (4 days postimmunization) is larger in terms of serum antibody (30–40-fold) than in terms of PFC or RFC (20- and 11-fold, respectively). A morphological study of RFC in nonimmunized mice showed that about 90% of rosettes were formed by small lymphocytes in both lines. The remainder were medium-sized lymphocytes. At the peak of the cellular response the RFC have differentiated into large lymphocytes, blast cells, and plasma cells. The contribution of plasma cells to RFC is much greater in the high than in the low line. The cytodynamic and morphologic results presented in this article are compatible with the hypothesis that the group of genes segregated in each line during the selective breeding control and regulate the rate of multiplication and differentiation of the antibody-producing cells. Footnotes Submitted: 14 December 1972

Ward, Peter A.; Cohen, Stanley; Flanagan, Thomas D.

doi: 10.1084/jem.135.5.1095pmid: 4623314

Infection of chick embryos wih either Newcastle disease virus or mumps virus and infection of BGM cell cultures with mumps virus result in the elaboration of chemotactic activity for neutrophils and macrophages. These factors cannot be found in lysates of uninfected cells. They do not appear to be associated with the viral particles per se, but rather are present in virus-free supernates from infected fluids. Ultracentrifugal studies of the neutrophil chemotactic activity in allantoic fluid of embryos infected with the two different viruses indicate a similar biphasic distribution of activity, while fluid from the mammalian cell cultures shows a single zone of leukotactic activity, further suggesting that the infected cell, rather than the virus, is responsible for the leukotactic activity. Virus-infected cells also release a substance(s) which is itself not leukotactic but which can interact with human C3 or C5 to generate such activity. This leukotactic factor-generating substance is similar to that reported in another virus-infected cell system. It is postulated that the leukotactic factors elaborated as a result of virus infection of cells may play a protective role in vivo. Footnotes Submitted: 3 January 1972

Lane, F. C.; Unanue, E. R.

doi: 10.1084/jem.135.5.1104pmid: 4623315

Spleen cells of mice infected with Listeria monocytogenes were adoptively transferred to normal mice. Such lymphocytes conferred resistance to a lethal challenge with Listeria . Hyperimmunization of the donor reduces the number of cells necessary to transfer effective immunity. Such spleen cells if treated with anti-θ serum do not transfer resistance to Listeria . Hence, thymus (T) lymphocytes are involved in the resistance to infection with the facultative intracellular bacteria L. monocytogenes . Footnotes Submitted: 23 January 1972

Atkins, Elisha; Feldman, Joseph D.; Francis, Lorraine; Hursh, Ellen

doi: 10.1084/jem.135.5.1113pmid: 4112261

Experiments have been carried out to investigate the possible role of the sensitized lymphocyte in mediating the fevers of delayed hypersensitivity. Rabbits were made delayed hypersensitive to one of several heterologous proteins (bovine gamma globulin, bovine serum albumin, or human serum albumin) by footpad injection of antigen or antigen conjugated with dinitrophenol and incorporated in complete Freund's adjuvant. At intervals after sensitization, various tissues were removed, and single cell suspensions were incubated overnight with either carrier protein or conjugate in vitro. Release of an endogenous pyrogen (EP) was assayed by intravenous injection of the supernatant fluid into unsensitized rabbits. Of the tissues tested only those containing both lymphocytes and pyrogen-producing cells, blood, spleen, and draining lymph nodes, released detectable amounts of EP when incubated with antigen in vitro. Incubation of normal blood cells with specifically sensitized lymphocytes and antigen also resulted in significant release of pyrogen. Similarly, blood leukocytes released EP in vitro after mixture with supernates derived from incubation of sensitized lymphocytes and antigen. Cells and supernatant fluids from draining lymph nodes were usually effective in activating normal blood leukocytes earlier after sensitization than were those from mesenteric lymph nodes, suggesting that such cells, or antigen, had migrated from the original site of sensitization. The activator was soluble, nonpyrogenic in the dosages tested, and required incubation of viable cells with specific antigen for its production. These properties suggest that it may belong to the class of "lymphokines," biologically active agents released from lymphocytes that have been activated by immunologic or certain nonimmunologic stimuli. Footnotes Submitted: 10 January 1972

Choi, Yong Sung; Good, Robert A.

doi: 10.1084/jem.135.5.1133pmid: 5022177

Synthesis and secretion of Ig by chicken lymphoid cells was studied. Both spleen and bursa cells synthesize and secrete IgM and IgG whereas Ig was not detected in thymus cells. In contrast to the spleen cells which synthesize H and L chains in balanced quantities, the bursa cells synthesize and secrete free L chains. In addition to the lymphoid cells which secrete IgM or IgG, the bursa appears to contain a cell population which synthesizes nonsecretory Ig. The structure of this Ig was studied by specific serological precipitation and by SDS-acrylamide gel electrophoresis. The H chains of this nonsecretory Ig are serologically related to µ-chains and exhibit a smaller molecular weight (i.e., ∼50,000) in SDS-acrylamide gel electrophoresis than H chains of IgG and IgM synthesized by the spleen cells (i.e., ∼70,000). Footnotes Submitted: 1 December 1971