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Using CT as the test antigen, we sought 1) to learn whether primary immunization at 1 mucosal site caused priming of distant nonstimulated mucosae, 2) to study the role of migrating memory cells in the dissemination of mucosal priming, and 3) to compare disseminated priming with priming that occurs at the site of initial immunization. CT given i.c. or i.d. caused priming in tracheal and nonexposed enteric mucosae; i.t. immunization, however, did not cause detectable enteric priming. Adoptive transfer of immune TDLs showed that priming was conveyed by migrating memory cells. These appeared to be of 2 types: those that recirculated briefly before settling in MALT, and those that continued to recirculate until recruited by antigen to the site of mucosal challenge. Both types were required for secondary responses at mucosae distant from the site of priming. The time-course of disseminated mucosal priming resembled that of priming at the site of initial CT exposure, both lasting at least 16 wk. Disseminated priming persisted better in jejunal than tracheal mucosa, suggesting that the subgroup of memory cells that did not continue to recirculate settled preferentially in jejunal MALT. Disseminated priming supported smaller challenge responses than priming at the site of initial CT exposure did, suggesting that sessile memory cells also contributed to the latter process. These observations extend the concept of a "common mucosal immune system" to include cellular dissemination of mucosal priming, but also show quantitative differences between local and disseminated priming that probably reflect the patterns of distribution of migrating and sessile memory cells.

The early work on lymphocyte recirculation assumed that all recirculating lymphocytes composed a common pool and that the composition of this pool could be inferred from studies on thoracic duct lymph. These propositions are examined in the light of more recent evidence, particularly from experiments on the traffic of lymphocytes through the lamina propria of the small intestine. There has been little speculation on the functional significance of lymphocyte recirculation apart from the suggestion that it increases the efficiency of regional immune responses by allowing antigen-induced selection of precursors from pool larger than that accommodated by the regional nodes alone. In addition, the mounting of a local immunity is dependent on a peripheral recirculation through the tissues, notably in the case of the secretory immune system of the intestine.

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.

The immune response has been reported to be modulated by sex hormones in several models, and estrogen receptors have been demonstrated in the human thymus. We therefore investigated the presence of estrogen and androgen receptors among human peripheral T cells; thoracic duct lymph provided large amounts of circulating lymphocytes. Pure T cells were obtained by negative selection by using complement-dependent cytotoxicity with a monoclonal antibody against a monomorphic determinant of class II histocompatibility antigen (HLA-DR). Furthermore, subsets of OKT8-positive and OKT8-negative lymphocytes were selected by using an OKT8-like monoclonal antibody. Sex steroid binding was determined on purified nuclei; no androgen receptors could be demonstrated on peripheral T cells. The cytoplasmic [3H] 17-beta-estradiol-receptor complex was always translocated to the nucleus in vitro within 1 hr at 37 degrees C; no estrogen receptors were demonstrable on purified OKT4-positive subsets. Assuming that estrogen receptors were evenly distributed among OKT8-positive cells, their level could be estimated to be about 40 fmol/mg DNA. The restriction of estrogen receptors to T cells bearing the "suppressor-cytotoxic" phenotype suggests a possible pathway for the modulation of T cell suppressive activities by estrogens.

The presence of lymphocytes with internal IgA among cells from rat thoracic duct lymph wdy. The number of cells detected was greater in animals kept in a convential animal house compared with those maintained under specific pathogen-free conditions. Thoracic duct lymph from B rats and adult thymectomized rats also contained cell with internal IgA. The surface Ig of the IgA-containing cells was studied using a double-labeling technique with (126I) anti-Ig to detect surface Ig, and fluorescein-conjugated anti-IgA in large amounts, but very little IgM and no surface IgG2. The surface IgA was not acquired passively.

We have examined rabbit lymphocytes from several tissue sources for membrane immunoglobulins with anti-antiserum for alpha, mu and iota heavy chains, as well as with an antiserum specific for a rabbit thymus lymphocyte antigen (RTLA). Among lymphocytes from bronchus-associated lymphoid tissue (BALT), Peyer's patches, thoracic duct and gut mucosa exclusive of Peyer's patches, alpha and mu predominated with roughly equal percentages of each being found. The distribution of cells with iota receptors was approximately half that of either of the two other classes. In splenic lymphocytes mu predominated, followed, respectively, in numbers by iota and alpha. Detection of RTLA on almost all thymus lymphocytes (94%), most thoracic duct lymphocytes (72%), and lower numbers of peripheral blood lymphocytes (44%), Peyer's patch lymphocytes (17%), and splenic lymphocytes (20%) indicated a similar distribution of this antigen between the lymphoid organs as has been found for the mouse thymus lymphocyte-specific antigens. Low numbers of RTLA-bearing lymphocytes were detected in both the BALT (18%) and the gut mucosa (11%). An unexpected finding was the number of "null" cells among the gut mucosa population, bearing neither thymic nor heavy chain markers. These results in toto support the view that lymphoid aggregates in lung and gut may both contain precursor populations of B cells destined for IgA production.

A comprehensive study of lymphocyte traffic in AO rats was performed under conditions as near to the physiological state as was practicable. In the light of previous results on the effect of environmental factors on the migratory behaviour of lymphocytes, thoracic duct cells were passed from blood to lymph in an intermediate rat before injection into a series of recipients for examination at time intervals from 1 min to 24 hr. At 1, 2 and 5 min after injection most of the labelled cells were in the blood, lungs and liver. The concentrations in these compartments fell over the next 25 min as the cells entered the spleen, lymph nodes (LN) and Peyer's patches according to a regular pattern. The peak localization in these latter organs occurred between 1 hr and 18 hr. Each organ had a characteristic time pattern of lymphocyte localization. Entry into mesenteric LN lagged behind other LN until 2.5 hr after injection following which mesenteric LN localization easily outstripped that in other LN to reach a delayed peak at 18 hr. Intravenously injected lymphocytes began to reappear in thoracic duct lymph in large numbers earlier than in previous studies so that the time taken for most T lymphocytes to cross LN from blood to lymph fell within the broad time band of 4-18 hr. Lymphocytes took on average 5-10 min to cross high endothelial venules when entering LN from the blood.

The primary migration of lymphocytes from the blood was compared in nude rats and in euthymic rats. The flatter endothelium in the post-capillary venules (PCV) in the lymph nodes of nude rats was as efficient as the high endothelium of PCV in euthymic rats at capturing both T and B lymphocytes from the blood, although lymphocytes took a longer time to cross the PCV wall in nude recipients. The organ distribution of both lymphocytes and lymphoblasts ([125I]UdR-labelled cells) was broadly similar in nude and euthymic recipients. A second aim was to compare B and T lymphocytes with respect to the rate and sites at which they leave the blood after intravenous injection. As judged by sampling venous blood, B lymphocytes left the blood faster, but this was partly attributable to a larger intravascular pool of B lymphocytes in small blood vessels, especially in the lung. Thoracic duct lymphocytes from nude rats collected under standard conditions (16 h, O degrees C) entered the cervical lymph nodes very poorly, but when lymphocyte transfer was performed under more physiological conditions entry of B lymphocytes into lymph nodes was about half that of T lymphocytes. B lymphocytes did show a slight preference for entry into Peyer's patches compared with lymph nodes.

Lymphocytes exchanged rapidly and extensively between mutually tolerant, allogeneic rats following surgical parabiosis. Tolerated allogeneic cells persisted for an extended period in parabiotic partners after separation. A rapid exchange of cells was also observed between parabiosed syngeneic pairs of normal and allograft tolerant rats. This was accompanied by early termination of the tolerant state manifested by skin graft rejection and return of specific lymphocyte alloreactivity.

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.

Thoracic duct lymphocytes from patients receiving thoracic duct drainage as a pretransplant therapy were examined for cell surface markers. Patients followed over the drainage time period showed a variable but decreasing percentage of E-rosette-positive cells in the lymph fluid. A substantial percentage of these E-rosette-positive cells also had C3 receptors on their cell surface. Reactions of the whole lymphocytes with a heteroantisera to human B-lymphocyte antigens reflected the increasing proportion of B cells in the samples, but also indicated that a fraction of the T cells have Ia-like antigens on their surface marker characteristics. Significance of these cells with respect to graft survival is discussed.

Human peripheral blood lymphocytes (PBL), obtained from patients with a variety of malignancies, when incubated in vitro with phytohemagglutinin (PHA), lysed fresh autologous tumors during a short-term 51Cr-release assay. These PHA-activated killer (PAK) cells produced maximum lysis of tumor cells by 4 to 8 hr of co-cultivation. PHA incubation induced the generation of cells lytic for autologous and allogeneic tumors but not autologous or allogeneic PBL. Cold target inhibition studies demonstrated that autologous and allogeneic tumors of various histologic types all shared the determinants recognized by PAK cells. Some adherent cells were necessary for generation of these PAK, but higher numbers were suppressive. Augmentation of tumor cell lysis was seen when adherent cells were partially removed before PHA activation. The PAK effector cell was OKT3+, OKT8+. The precursor cell of the PAK was separated from natural killer (NK) cells on Percoll gradients and was generated from thoracic duct lymphocytes, a population devoid of NK cells. Furthermore, the phenotype of the majority of the precursor cells was OKT3+, OKM1-. Activation by PHA for 2 days was found to be an efficient and convenient method for generating lymphoid cells lytic for fresh autologous human tumor. The biologic and possible therapeutic role of these cells is currently being investigated.

Endothelial cells from the canine or human thoracic duct were harvested using 0.2% collagenase digestion and grown in Media 199 supplemented with fetal bovine serum. The canine endothelial cells grew to confluence (4.4 to 12 X 10(4) cells/cm2) in 6 to 10 d; doubling times ranged from 1.5 to 2.8 d. There was a minimum critical density for cell growth between 5000 and 10 000 cells/cm2. The canine endothelial cells have been maintained in culture for periods up to 11 mo. The human thoracic duct endothelial cells are more difficult to grow and maintain. Endothelial cells were isolated from 5 out of 35 human thoracic ducts and grew for periods of up to 2 wk before degenerating. Both human and canine endothelial cells were Factor VIII positive. It has thus been demonstrated that it is possible to grow canine and, less easily, human thoracic duct endothelium in tissue culture.

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.

When thoracic duct lymphocytes (TDL) or mesenteric lymph node (MLN) cells from mice primed 1 day before with either sheep erythrocytes (SRC) or horse erythrocytes (HRC) were transferred together with both SRC and HRC to irradiated mice, antibody responses measured 7 days later could not be detected to the priming antigen but were high to the other antigen. Furthermore, this unresponsiveness of TDL and MLN to the priming antigen could not be abrogated by delaying antigen challenge of the transferred cells for 1-2 wk. Previous work had shown that short-term priming with antigen also induced specific unresponsiveness in spleen cells on adoptive transfer. Unresponsiveness in these cells, however, was only of temporary duration, full recovery in the reactivity of the cells being observed when challenge with the priming antigen on transfer was delayed for 5 or more days. Since the present work showed that such recovery from initial unresponsiveness on transfer was unique to spleen cells and did not apply to TDL or MLN, it appeared that different mechanisms were responsible for the unresponsiveness in the three populations. It is proposed that the unresponsiveness detected in TDL and MLN cells in the present study resulted from a deficiency of antigen-reactive cells, these cells having been recruited to the spleen, i.e., a region of antigen concentration. This concept of antigen-induced selective recruitment of circulating lymphocytes was supported by evidence that (51)Cr-labeled heterologous erythrocytes indeed localized largely in the spleen after intravenous injection but not in MLN.

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.

A flow cytophotometry method has been evaluated for the enumeration of rat lymphocyte subpopulations in lymph and lymph nodes. The cells were prepared by incubating them with complement and anti-Ig or anti-T antiserum, followed by trypan blue staining. Cytophotometry was used for automatic registration of dead and viable cells. Parallel viability counts were done in a microscope. In addition the cytotoxic activity of the anti-Ig antiserum was compared with its surface binding capacity in a direct immunofluorescence assay. Correlation was found between the automatic and microscopical registrations.

Studies of the migration of antigen-specific regulatory T cell subsets responding to gut immunization were undertaken to clarify their migratory potential and the role of antigen in their localization. In initial experiments, lymphocytes collected from the thoracic duct of rats after immunization of Peyer's patches (PP) with keyhole limpet hemocyanin (KLH), were enriched for T helper (Th) cells and labelled with the fluorochrome H33342. In other experiments, a higher frequency of antigen-specific T cells was achieved by short-term culture of the enriched Th cells in the presence of KLH and the blast cells labelled with 3H-thymidine. The distribution of both populations was determined after injection into immunized and unimmunized syngeneic recipients. Whereas the uncultured population (predominantly small Th cells) localized almost exclusively in follicular lymphoid tissues, the cells expanded by secondary culture (predominantly Th blasts) appeared in the gut lamina propria (LP) initially, then in PP and mesenteric lymph nodes. The Th blasts in the LP were almost always seen in close proximity to the gut epithelium. However, the migration of neither population appeared to be influenced significantly by antigen, in contrast to previous findings with regard to IgA-committed B cells. The initial subepithelial location of Th blasts in the gut LP and their subsequent appearance in PP may provide a mechanism by which antigen presented by epithelial cells could influence B cell differentiation in PP through modulation of signals expressed by these T cells.

Thoracic-duct cannulation of mesenteric lymphadenectomized (MLNx) congenitally athymic nude rats was studied as a method of obtaining peripheral lymph cells. A higher recovery of non-lymphoid cells (NLC) was obtained from nude than from euthmyic littermates. Both a higher percentage and a greater number of NLC were found in nude animals. Most of these cells resembled dendritic or veiled cells and were strongly positive for Ia antigens. This population could further be enriched by irradiation of the animal, but with a risk of cell damage. Splenectomy had no effect on early output of Ia+ NLC. A substantial population of lymphoid cells from MLNx nude rats expressed T-cell antigens defined by the monoclonal antibodies OX 19, OX 8 or W3/25. These cells were more radiosensitive than were mature T cells. In addition, a large population of cells in the peripheral lymph from nude rats did not display surface antigens recognized by monoclonal antibodies directed either against B cells or against T cells. This cell fraction increased after irradiation. These cells resembled small lymphocytes but had a more irregular nucleus and multiple large granules.

The frequency of lipopolysaccharide (LPS)-reactive B cells developing into clones that secrete the various immunoglobulin (Ig) classes has been determined in vitro, in cells from BALB/c mice, under culture conditions which detect all growth-inducible cells. Secretion of the different Ig classes was assessed in the protein A plaque assay for Ig-secreting, plaque-forming cells by using developing antisera specific for either IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA. In all lymphoid organs tested (spleen, bone marrow, mesenteric lymph nodes and thoracic duct), a considerable proportion of all B cells (5-20%) was induced by LPS to yield a clone of IgM-secreting cells. Frequency determinations of LPS-reactive cells giving rise to descendants secreting other Ig isotypes revealed that, on an average, and irrespective of the origin of the cells, 7% of all IgM-secreting clones switched to the synthesis of IgG1, 39% to IgG2, 41% to IgG3 and 1% to IgA. Roughly the same frequencies of B cells switching CH gene expression were found among spleen cells of athymic nude mice. No correlation was found between the clonal frequencies of CH gene expression in polyclonally activated B cells and the in vivo "background" Ig-secreting cells suggesting that the CH gene expression in B cells is influenced by the quality of stimulation and other regulating influences.

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.

Thoracic duct and spleen cells of normal (unimmunized) adult rats were fractionated according to size by 1 times G velocity sedimentation. Fractions were tested for their ability to restore the adoptive antibody response of irradiated hosts to horse spleen ferritin. A constant source of T cells (small numbers of unfractionated thoracic duct cells) was added to each fraction in order to monitor the B cell activity of the latter. Although large and small cell fractions of the spleen showed restorative activity, only the small cell fractions of the thoracic duct lymph showed activity. The turnover rate of the spleen cell fractions was determined by treating donors with high specific-activity 3H-thymidine for 48 hr before splenectomy. Rapidly dividing cells are preferentially killed by this treatment. The results suggest that a considerable proportion of large, intermediate, and small virgin B cells turn over within 48 hr. The cell surface of the various spleen cell fractions was examined for the presence of immunoglobulin (Ig) and a receptor for complement. The percentage of Ig-bearing cells in the large cell fractions was similar to the percentage of cells bearing IgM and a receptor for complement. However, the majority of Ig-bearing cells in the small cell fractions did not show the latter two surface markers. Experiments with the fluorescence-activated cell sorter showed that the large functionally active B cells bore surface IgM. The experimental findings suggest that there are subpopulations of virgin B cells in the spleen of the adult rat which differ with respect to size, migration pattern, turnover rate, and cell surface characteristics. The relationship of these cells to one another is discussed in the framework of an antigen-independent model of B cell maturation in the rat.

The T lymphocyte-deprived (B) rat, produced by X-radiation and bone marrow reconstitution of adolescent thymectomized animals, exhibits a true immunological deficit and are unable to reject histoincompatible heterotopic cardiac allografts. A comprehensive survey of lymphocyte traffic in B recipients was performed to correlate the differential potency of specifically sensitized lymphocyte populations mediating re-establishment of immune responsiveness toward the graft, with their migratory and recirculatory behavior. 111In-oxine-labeled thoracic duct lymphocytes (TDL) were retained in the peripheral blood and migrated from nonlymphoid organs to lymph nodes of B recipients in higher proportion than any other lymphoid population, particularly splenic lymphocytes (SL). Although all cell groups but TDL were sequestered in the spleen in equal and relatively large numbers, no differences were found between the lymphocyte populations tested in their capacity to accumulate in the grafts. In contrast, an increased avidity in the allograft of 125IUdR-labeled TDL and lymph node (LNL) lymphoblasts, as compared to 125IUdR-labeled SL, resembles closely the results of functional studies of the differential potency of adoptively transferred cells. We assume that specific cellular interactions induced by the accumulated 125IUdR-labeled cells invoke nonspecific mechanisms for the recruitment of other uncommitted 111In-labeled lymphocytes which recirculate between blood and lymph and localize indiscriminately in the allograft amplifying its rejection. The latter lymphocytes can be "armed" by adherent cells residing in the lymphoid organs of graft recipients, particularly spleen, and subsequently increase the penetration of the foreign tissue. When radiolabeled lymphocytes were traced in B recipients experiencing rejection of their allografts following transfer of sensitized cells plus lymphokine, their migration patterns as well as blastogenic response in B hosts were similar to those observed during acute rejection of cardiac allografts in unmodified hosts. Thus the similarities between the rejection network brought by alloimmune cells into otherwise unresponsive animals and immunocompetent animals able to reject their grafts are stressed.

Three uniform populations of T and B cells exposed to varying amounts of x-irradiation are examined utilizing computer-assisted morphometric analysis. These populations are: thoracic duct lymphocytes (TDL) from congenitally athymic (nude) mice (B cells); TDL from CBA mice treated with anti-Ig plus complement (T cells); and computer-selected untreated T cells from CBA TDL. Irradiated B cells show a more even dispersion of the nuclear chromatin and a dose-dependent increase in relative nuclear area beginning with the lowest dose evaluated (50 rads); no significan change in total optical density (OD) is demonstrable over the dose range evaluated (0 to 2000 rads). Anti-Ig-treated irradiated T cells demonstrate an initial shift toward lower OD values as a function of dose followed by a marked rise of OD values at 2000 rads, where numerous densely staining Feulgen-positive aggregates are identified. The relative nuclear area of this cell population also shows a biphasic response to radiation injury with an initial increase at the lower dose levels followed by a progressive decline to approximate control levels at 2000 rads. This effect is mirrored by the alteration in total OD which, after a decrease at low dose levels, approximates control values at 2000 rads. The computer-selected T cells show little change in OD values at the low-dose levels but show a marked increase in the more densely staining Feulgen-positive material following 2000 rads. This population reveals no apparent change in either relative nuclear area or total OD as a function of dose. Thus, untreated computer-selected T cells exhibit remarkably little evidence, morphologically, of radiation injury of doses associated with pronounced alterations on the part of B cells. In addition, treatment of a mixed cell population (CBA TDL) with anti-Ig plus complement to remove the B cells appears to alter the response of the residual T cells to radiation injury. These results, in conjunction with recent evidence to support the concept that T cells possess surface Ig, suggest that an Ig-anti-Ig interaction may alter the radiosensitivity of T cells.

A series of 15 patients was treated for chylothorax over a 20-year period. The anatomy, physiology, and diseases of the thoracic duct are described, and a plan for the management of chylothorax is presented. If conservative therapy (e.g., aspiration or drainage with restriction of oral intake and intravenous replacement) is not successful after two to three weeks, surgical treatment is necessary and efficacious. The thoracic duct is explored by a full thoracotomy on the side of the effusion. It is readily seen if 6 to 8 oz of a mixture of milk and cream is given to the patient a few hours before operation. The milky fluid drips from the open duct, which is easily oversewn.

The migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T- and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with 3H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.

Wistar rats immunized with irradiated syngeneic methylcholanthrene-induced sarcoma, Mc7, are able to suppress a subsequent challenge of this tumor. Cells obtained from thoracic-duct lymph of these tumor-immunized animals and infused intravenously into normal syngeneic rats transfer specific tumor resistance to the recipients. These mediators of tumor resistance have the characteristics of T lymphocytes that are in a nonproliferating state and have an effective life span at least 2 weeks in normal recipient rats.

A technique for the collection of lymph from the lacteals of the small intestine of the rat is described. The output of lymphocytes from an area possessing a Peyer's patch is 10 times greater than in lymph from an area lacking one, but the proportion of lymphocytes with surface immunoglobulin (sIg), surface immunoglobulin A (sIgA), and internal immunoglobulin A (iIgA) are similar in the 2 areas. A lower percentage of lymphocytes bear sIg in lymph afferent to the mesenteric lymph gland compared with that found in the thoracic duct. However, a higher proportion of these lymphocytes bearing sIg have IgA on their surface in lacteal lymph compared with thoracic duct lymph. The reasons for these differences between the lacteal lymph and thoracic duct lymph are discussed.

Results obtained using negative selection protocols indicate that it is possible to obtain vaccinia-virus-specific killing of cells expressing H-2 types other than those encountered during physiologic development in thymus. This response has been found to be neither reciprocal for different H-2 types, nor identical for influenza and vaccinia viruses. Prior priming with virus apparently suppresses the capacity of T cells to be sensitized to H-2-different virus-infected cells.

In order to differentiate between donor (chimeric F1) cells and host cells as being responsible for suppression in transplantation tolerance (TT), cells from tolerant donors were first subject to negative selection through F1 hybrid intermediate host animals. This revealed that the suppressor cells was neither completely removed from the lymph nor recoverable from the lymphoid tissues of the filter rat when highly suppressive inocula were used, suggesting that suppression did not directly depend on cells with receptors for alloantigens. The phenotype of donor and host cells in the recirculating pool was studied with fluorescent antisera and showed that both host cells and chimeric F1 cells were in the thoracic duct lymph of tolerant rats and were capable of recirculation. T and B lymphocytes of both types were present but the IgG-positive, presumptive memory B cells were highly enriched in those rapidly recirculating cells, obtained by filtration of tolerant inocula through irradiated intermediate hosts. These cells were also highly enriched for suppressor function. Methods which selectively depleted either the chimeric T cells or the B cells were applied to tolerant inocula and on adoptive transfer of these inocula, suppression was eliminated only when chimeric T cells were eliminated. This strict dependence of suppression of F1 hybrid T cells is interpreted as evidence that these cells probably suppress directly, via an anti-idiotypic mechanism, the alloreactive cells bearing idiotype-positive major histocompatibility complex receptors.

The lack of progress in development of vaccines to stimulate local protection in the urinary tract is attributable in part to the lack of information regarding mechanisms of local immunity and to the difficulty in antigen administration at this site. Experiments reported in this paper indicate that the urinary tract in rats forms part of an effector network linking mucosal organs (the common mucosal immune system). Concomitant immunization of the intestine and urinary tract with ovalbumin resulted in the appearance at both sites of antiovalbumin-containing cells (ACC) of IgA specificity. The gut origin of ACC in the urinary tract was confirmed by demonstrating in similarly immunized rats abrogation of the urinary tract response by chronic drainage of the thoracic lymphatic duct during the post-challenge period and by demonstrating the appearance of radiolabelled ACC in the urinary tract after injection of labelled autologous thoracic duct lymphocytes collected during the post-challenge period. These experiments indicate a role for oral immunization in enhancing the local antibody response in the urinary tract against invading pathogens.

Resistance to a lethal challenge with Theileria parva (Muguga) was transferred between two pairs of chimeric bovine co-twins with 5 to 9 X 10(10) syngeneic thoracic duct leucocytes from the immunised partner. In one recipient, the infection became established and was eliminated. In the second recipient, the disease did not become patent. Both recipients were immune to a second challenge of T parva (Muguga). The results confirm both that chimeric twin calves are suitable as a model for the adoptive transfer of cell-mediated immunity in cattle and that recirculating lymphocytes confer resistance against infection with T parva to immune cattle.

The corticosteroid effect on circulating lymphocytes in a steroid-sensitive species (rat) was studied by immunofluorescence. Both normal and neonatally thymectomized animals were injected with a high dose of prednisolone. T- and B-lymphocytes as well as T-lymphocyte subsets were analyzed in thoracic duct lymph during the involution phase (3 h after injection) and the restitution phase (17 h after injection) using conventional and monoclonal antisera. Neonatal thymectomy significantly reduced the number of circulating T-helper and T-non helper cells. In the involution phase, after a corticosteroid injection, there was a 64% reduction of lymph cell content in normal rats, affecting both T- and B-lymphocytes. Thymectomized animals showed an even more pronounced disappearance of cells. Counted in percentage, more B- than T-lymphocytes disappeared from the circulation. The T-non helper cells disappeared to a greater extent than the T-helper cells did. Cells with Ia-antigen showed about the same values as the B-lymphocytes. During the restitution phase all cells types were restored to normal levels. It seems that a high prednisolone dose has a more pronounced effect on the circulation of B- than of T-lymphocytes, and the T-non helper cells are probably more sensitive than the T-helper subpopulation. These data support the hypothesis that a major corticosteroid effect is a trapping and redistribution of circulating lymphocytes, more pronounced for certain cell types.

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.

Highly purified populations of C57BL/6 (B6) L3T4+ and Lyt-2+ T cell subsets were compared for their capacity to exert alloreactivity to class I vs. class II H-2 differences in vivo. B6 Lyt-2+ cells responded strongly to the class I different mutant, bm1, as manifested by DNA synthesis in the spleen of irradiated mice followed by entry of blast cells into thoracic duct lymph, induction of splenomegaly in newborn mice, production of lethal GVHD in irradiated mice, and skin allograft rejection. By all of these parameters, B6 Lyt-2+ cells showed almost total unresponsiveness to the class II-different mutant, bm12. Reciprocal results were observed with B6 L3T4+ cells, these cells responding strongly against bm12 but not against bm1. In the case of purified T cell subsets from other strains, CBA/Ca and B10.BR L3T4+ cells both responded well to a full H-2 difference. Responses by Lyt-2+ cells from these strains were weaker, especially for CBA/Ca cells. The implications of these findings are discussed.