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Thoracic duct lymphocytes from pregnant PVG rats differed markedly from similar cells collected from non-pregnant donors in their reactivity in popliteal lymph node assays performed in (PVG x DA) F1 hybrid hosts. Cells from pregnant rats failed to evoke an increase in popliteal node size commensurate with higher challenge doses of lymphocytes. This modification in maternal lymphocyte reactivity was not dependent on the presence of foetuses genetically compatible with the F1 hybrid rats used in the popliteal lymph node assay. The mechanism responsible for the decrease in size of responding F1 hybrid popliteal lymph nodes appeared to be based on a reduced capacity of lymphocytes from pregnant donors to stimulate a response by host lymphocytes.

Rat spleen cells are shown to be unresponsive to sheep red cells (SRBC) in vitro under conditions in which thoracic duct lymphocytes (TDL) respond very well. By adding unresponsive spleen cells to responsive TDL cultures, the spleen cells are shown to contain an inhibitor capable of preventing the response to SRBC. The inhibitory activity is a property of live cells; it is sensitive to radiation doses as low as 100 R x rays and to mitomycin C. It can be completely removed from spleen cell suspensions by extraction with large amounts of carbonyl iron or by filtration through nylon wool columns. It is less efficiently removed by filtration through Sephades G-10 columns, and is completely resistant to the cytotoxic effects of silica. From a practical point of view, extraction of a spleen cell suspension with carbonyl iron is a useful method of obtaining fully responsive lymphocyte populations from rat spleen.

In mixed leucocyte culture (MLC), using human spleen cells or thoracic duct lymphocytes, antibody secretion was induced, measured as plaque-forming cells (PFC) in a haemolysis-in-gel assay with fluorescein isothiocyanate (FITC)-coupled sheep erythrocytes (SRBC) as targets. Peak antibody secretion was seen on day 5. Using protein-A-coupled SRBC as targets and developing antisera, antibody secretion in MLC was found to be of IgM, IgG and IgA type. There was no correlation between the number of PFC against FITC-SRBC in MLC and DNA synthesis. Supernatants from MLC failed to induce antibody secretion.

When thoracic duct lymphocytes (TDL) from mice given sheep erythrocytes (SRC) 1 day previously (1-day-primed TDL) were placed in microcultures with antigen for 5-6 days, the cells failed to produce antibody to SRC, but responded well to horse erythrocytes (HRC); reciprocal results were obtained with TDL from HRC-injected mice. The specific unresponsiveness of the TDL was observed despite the presence of an allogeneic factor in the cultures; this factor exerted not only a macrophage-like effect (TDL from normal mice failed to respond in the absence of this factor) but also a T-cell-replacing function. It was concluded therefore that the unresponsiveness of the TDL was the result of selective recruitment of specific B lymphocyte precursors from the recirculating lymphocyte pool to the lymphoid tissues; whether there was also recruitment of specific T lymphocytes was not investigated. Since addition of 1-day-primed TDL to cultures of normal TDL did not inhibit the response of the latter, there was no evidence that active suppression accounted for the unresponsiveness. The spleen appeared to be the main site for recruitment since 1-day-primed spleen cells placed in microcultures contained above normal numbers of specifically-reactive precursors. This was in striking contrast to the effect of transferring 1-day-primed spleen cells in vivo where, as previously reported, the cells fail to respond to the injected antigen within 1 wk of transfer. An explanation for this paradox is discussed.

The migration pattern, tissue distribution, and turnover rate of unprimed and primed B lymphocytes involved in the adoptive anti-DNP response was studied. The adoptive primary response restored by unprimed spleen or thoracic duct cells passaged through an intermediate host (intravenous injection and subsequent collection in the thoracic duct lymph) was markedly diminished as compared with that restored by unpassaged cells. On the other hand, the adoptive response restored by passaged spleen or thoracic duct cells from DNP-primed donors was greater than or the same as that restored with unpassaged cells, respectively. This suggests that unprimed B cells change from nonrecirculating to recirculating lymphocytes after exposure to antigen. Studies of the adoptive anti-DNP response restored by unprimed or primed bone marrow cells showed little change in the time-course or amplitude of the response restored by either population of cells. The relative inability of marrow cells to carry immunological memory was related to the inability of recirculating memory cells to penetrate the marrow. The turnover rate of unprimed and primed B cells was investigated by treating the cell donors with [(3)H]thymidine for 48 h before removal of thoracic duct or spleen cells. The adoptive anti-DNP response restored by unprimed or primed cells was not affected by [(3)H]thymidine treatment. This indicates that both populations of cells turn over slowly. However, our previous studies show that unprimed B cells involved in the adoptive antibody response to ferritin turn over rapidly. The different findings are discussed in the context of antigen-dependent B-cell maturation.

Thoracic duct lymphocytes from rats primed with DNP-BGG confer on irradiated, syngeneic recipients the ability to mount a large, IgG anti-DNP response after challenge with the immunizing conjugate. An analysis of this adoptive response lead to the following conclusions. the B memory cells which generate the IgG response carry IgG on their surface and are present in a subpopulation amounting to less than l0% of all B cells in thoracic duct lymph. The view that memory cells carry exclusively IgD is untenable. The majority of B lymphocytes in thoracic duct lymph are recirculating cells which carry surface IgM...

We have previously demonstrated that interferon (IFN) transiently inhibits the number of recirculating thoracic duct lymphocytes (TDL), while other researchers have demonstrated similar decreases in lymphocytes in peripheral blood. In the present study we examined whether or not this decreased lymphocyte recirculation contributes to IFN's suppressive effects on antibody formation. Immunizing at varying times during the period of maximal decrease in lymphocyte recirculation, there are no significant differences in the number of plaque-forming cells (PFC) or antibody titers generated to either a T-dependent antigen, sheep erythrocytes (SRBC) or a T-independent antigen, Type III pneumococcal polysaccharide (SSS-III). These results suggest that: (i) the suppressive effects of IFN on antibody responses are not related to changes in lymphocyte recirculation and (ii) lymphocyte circulation is not required during the early phases of the humoral immune response to antigens.

Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.

Thoracic duct cells and spleen cells were tested for their ability to restore the primary antibody response of X-irradiated rats to bovine serum albumin (BSA), sheep red blood cells (SRBC), horse spleen femtin (HSF), and Salmonella typhi flagella. Spleen cells were at least as efficient as thoracic duct cells in restoring the response to BSA, HSF, and Salmonella typhi flagella. In further experiments thoracic duct cells lacking large dividing lymphocytes were tested for their ability to restore the primary response. Large lymphocytes were eliminated by the in vitro incubation of thoracic duct cells for 24 hr at 37 degrees C or by treatment of thoracic duct cell donors with the mitotic inhibitor vinblastine sulfate 24 hr prior to cannulation of the thoracic duct. Experiments with SRBC show that incubated cells and cells from vinblastine-treated donors are as efficient as normal cells in restoring the primary antibody response. On the other hand, experiments with HSF and Salmonella typhi flagella show that incubated cells and cells from vinblastine-treated donors are about five times less efficient than normal cells in restoring the response. Normal thoracic duct cells were more efficient than incubated cells but less efficient than cells from vinblastine-treated donors in restoring the early response to BSA. The experimental findings indicate that the classes of thoracic duct lymphocytes which initiate the primary antibody response to SRBC differ from the classes which initiate the response to HSF and Salmonella typhi flagella, or BSA.

After intravenous injection into rats, both the attenuated strain R1Rv and the virulent strain H37Rv of Mycobacterium tuberculosis grow in the liver and spleen. However, the infected rats mount a specific immune response with great rapidity, giving a false impression of natural resistance to the tubercle bacillus. Adoptive immunity to tuberculosis was achieved by transferring thoracic duct cells from immunized donors to normal syngeneic recipients. The transferred immunity was vested in a population of lymphocytes uncontaminated with macrophages. The adoptive immunity was effectively expressed against both attenuated and virulent tubercle bacilli, and it was shown to be immunologically specific. Lymphocytes which conferred immunity to tuberculosis were not protective against Listeria monocytogenes infection, and vice versa. Immunity could not be transferred with either normal thoracic duct lymphocytes (TDL), heat-killed sensitized TDL, or serum from specifically immunized donors. The ability of TDL from BCG-immunized donors to confer immunity was maintained at an unimpaired level for at least 3 months after immunization.