Thymus-independent (B) cell proliferation in spleen cell cultures of mouse radiation chimeras stimulated by phytohemagglutinin or allogeneic cells.

Spleen cell cultures of radiation chimeras (thymectomized, lethally irradiated mice repopulated with bone marrow cells and thymocytes bearing different chromosomal markers) were stimulated by phytohemagglutinin (PHA) and F(1) allogeneic spleen cells. Karyotypic analyses showed a marked predominance of T mitoses on the 2nd and 3rd days of culture followed by a strong predominance of B mitoses on the 4th and 5th days. Analysis of cells undergoing their first mitoses showed that the majority of T mitoses on day 3 resulted from continuous T cell division, and that most cells entering their first mitoses at that time were of B type. Mixed lymphocyte cultures (MLC) of chimeras immunized against allogeneic spleen cells showed sometimes, but not always, a response different from "primary" MLC, with an earlier and stronger predominance of BM mitoses. The role of stimulated T cells in the induction of B mitoses was shown by (a) the incapacity of T-depleted spleen cells to be stimulated by PHA or in primary or secondary MLC, and (b) the restoration of the mitotic response of B cells to PHA by adding to the T cell-depleted culture either a very small number of T cell (identified by their different karyotype: "in vitro chimeras") or the cell-free supernatant of a 24 hr MLC.

Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex.

Conditions influencing Ig secretion by plasma cells have been studied with suspensions of murine plasma cells and myeloma cells by determining the release of (3)H-Ig after a pulse of biosynthetic labeling with L- [4,5-(3)H]-leucine. Ig secretion is insensitive to a variety of hormones, mediators, cyclic nucleotide derivatives, extracellular calcium depletion, and agents acting on mierotubules or microfilaments; i.e., to a number of factors which are involved in the regulation of secretion by cells with a storage compartment. On the other hand, Ig secretion is markedly inhibited by conditions which (a) lower intracellular calcium levels (ionophore A 23187 in Ca(++)-free medium), (b) induce partial sodium/potassium equilibration (the ionophores monensin and nigericin and, in the case of myeloma cells, ouabain and incubation in K(+)-free medium) or (c) uncouple oxidative phosphorylation. The first two situations are accompanied by striking alterations of the ultrastructural appearance of the Golgi complex, different in each case. These ultrastructural observations, together with autoradiographic experiments after a short pulse with L-[4,5-(3)H]-leucine, have led to the following hypothesis: (a) under Ca(++) depletion (3)H-Ig passes to Golgi vesicles but these vesicles are incapable of fusion or migration and therefore accumulate in exaggerated numbers in the Golgi area; (b) under partial Na(+)/K(+) equilibration, (3)H-Ig passes to Golgi vesicles which have an exaggerated tendency to fuse with other Golgi elements, thereby generating large vacuoles which store increasing amounts of Ig; (c) under energy block, multiple membrane fission and fusion events are inhibited and there is therefore, little intracellular transport of (3)H-Ig or alteration of cell ultrastructure.

Thymic and extrathymic origins of gut intraepithelial lymphocyte populations in mice.

We have investigated the origin of intraepithelial lymphocytes (IEL) populations in the murine gut, using reconstitution experiments in which the presence of thymus-derived cells of host or donor origin is rigorously controlled: RAG-/- mutant mice which have no T cells, were injected either with the bone marrow (BM) cells of nude mice or with selected peripheral lymph node (LN) T cells of euthymic mice. In thymectomized RAG-/- mice, injection of BM cells from nude mice led, after 2 mo, to the development of a peripheral B cell compartment and to the appearance, in the gut, of IEL bearing homodimeric CD8 alpha chains and either gamma/delta or alpha/beta TCR. In RAG-/- mice with a thymus, a similar injection led to complete lymphoid reconstitution, with the additional appearance in the gut of CD4+, CD8 alpha/beta+ or CD4+CD8 alpha/alpha+ IEL, all bearing alpha/beta TCR. In contrast, injection of LN T cells into these mice reconstituted a gut IEL population made of CD4+, CD8 alpha/beta+, or CD4+ CD8 alpha/alpha+ cells, all bearing alpha/beta TCR; CD8 alpha/alpha+ TCR-gamma/delta+ or alpha/beta+ IEL were not observed. These results demonstrate that the thymus and/or thymic-derived peripheral T cells are absolutely required for the generation of CD4+, CD8 alpha/beta+, and CD4+CD8 alpha/alpha+ IEL, which are thus thymus dependent. In contrast, TCR+ CD8 alpha/alpha+ IEL appear in the absence of the thymus, and thus are thymus independent.

The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta + lymphocytes reveals a major extrathymic pathway of T cell differentiation.

Gut intraepithelial lymphocytes (IEL) contain two independent T cell receptor alpha/beta + T cell populations, with different V beta repertoires. In DBA/2 mice (Mlsa, IE+), the CD4+ and heterodimeric alpha/beta CD8+ thymodependent T cell pool shows the same deletion of V beta 6, 8.1, and 11+ cells as found in peripheral lymphoid organs. In contrast, such deletions are not observed in the pool of IEL bearing homodimeric alpha CD8+ chains, in which these V beta families are frequently observed in high amounts. The size of this gut homodimeric alpha CD8+ IEL pool and its different V beta repertoire selection demonstrate the existence of a major extrathymic pathway of T cell differentiation with a gut-restricted localization. The large amount of the thymo-independent, homodimeric alpha CD8+ IEL found in the small bowel may contribute to a first line of defense against exogenous superantigens.

Specificity of the antibodies produced by single cells following immunization with antigens bearing two types of antigenic determinants.

A combination of double immunofluorescent technique and radioautographic localization of radioactive antigens was used to investigate the question whether single antibody-producing cells can make antibodies of more than one specificity after immunization with antigens bearing more than one determinant. When guinea pig gamma(2)-globulins, containing the F(ab')(2) and Fc determinants, were used to immunize rabbits, a small percentage of cells (3.7%) were stained with both the anti-F(ab')(2) and anti-Fc reagents. These results were shown to be due to the lack of absolute specificity of the detecting antigen and antibody reagents which can be obtained for use in this system. However, when immunological systems such as hapten-protein conjugates were used, and where completely specific antigen and antibody reagents could be prepared, the results were unequivocal. The individual lymph node cells from rabbits or guinea pigs immunized with hapten-protein conjugates produced antibodies against the hapten or antibodies against the antigenic determinants of the carrier molecule, never antibodies against both specificities.

The distribution of large dividing lymph node cells in syngeneic recipient rats after intravenous injection.

The distribution of large dividing lymph node or thoracic duct lymph cells, labeled in vitro with (3)H-thymidine, was studied in syngeneic recipient rats after intravenous injection. In most experiments the donor rats had been immunized with Bacillus pertussis 4 days earlier, but in some instances cells from nonimmunized donors were used. In smears, the labeled donor cells had the appearance of large lymphocytes or large pyroninophilic cells. By electronmicroscopy, the majority of labeled donor cells were seen to have only scanty endoplasmic reticulum. It was found that the labeled cells rapidly "homed" to lymphoid tissue and recirculated in the recipient, in a fashion resembling that of small lymphocytes. However, the distribution of labeled cells was found to depend upon the source of the donor cells. Cells from mesenteric lymph nodes or thoracic duct lymph showed a marked preferential accumulation in lymphoid tissue within or adjacent to the intestine, whereas cells from peripheral nodes accumulated preferentially in peripheral lymph nodes. Cells from any of these sources showed an equal tendency to accumulate in the white pulp of the spleen. Suspensions of small lymphocytes, labeled in vitro with (3)H-uridine, did not display a similar tendency to localize preferentially in lymphoid tissue in certain regions. It was also found that large dividing lymph node cells from donors immunized with an antigen (2,4-dinitrophenyl-bovine gamma globulin (DNP-BGG) or B. pertussis) showed a greater tendency to accumulate in a recipient lymph node containing that antigen than in the contralateral node. It was not determined whether the selective accumulation of large dividing lymphoid cells from different sources in lymphoid tissue of different regions in recipients was due to an antigen recognition mechansim or was the result of two different populations of cells with different "homing" mechanisms.

In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence.

Peanut lectin (PNL) binds to a majority of mouse thymocytes (Thc) in suspension. By using cell affinity chromatography on a column of anti-PNL antibody, Thc populations at least 96 percent pure in PNL + or - cells, as judged by immunofluorescence, were obtained. PNL(+) cells are rich in Thy 1 and poor in H(2) antigens, cortisone sensitive, unresponsive to phytohemagglutinin (PHA), and immunologically incompetent, as judged by mixed lymphocyte reaction, popliteal lymph node graft-versus-host assay, and by testing helper activity in a primary in vitro antibody response to sheep erythrocytes; the converse is true of PNL(-) cells. Thus, PNL(+) and (-) cells appear to correspond to cortical and medullary Thc, respectively, as previously suggested. In culture, PNL(+) Thc show poor viability and a weak proliferative response to concanavalin A (Con A), except when supernate (SUP) of 24 h Con A stimulated lymph node lymphocyte cultures, or irradiated lymph node cells, are added, in which cases a strong proliferative response to the mitogen is observed. A variety of control experiments showed that the proliferating cells did not result from preferential stimulation of a few contaminating PNL(-) Thc present in the PNL(+) Thc cultures. The blasts resulting from PNL(+) Thc proliferation display mitogen-induced cytotoxicity, and give rise to a population of medium-sized lymphocytes, mostly PNL(-), poor in Thy 1 and rich in H(2) antigens, PHA responsive, and immunologically competent in the above-mentioned assays. Fresh PNL(+) Thc responded in mixed lymphocyte reaction in the presence of SUP (lectin depleted) and since incubation in SUP alone did not confer reactivity on PNL(+) Thc, it appears therefore that (a) immature Thc possess alloantigen and mitogen-specific surface receptors but lack the capacity to respond by proliferation to receptor triggering without the help of extracellular factor(s) released by mature lymphoid cells stimulated by mitogens (b) cell division is associated with the acquisition of immunological responsiveness, characteristic of mature T lymphocytes. The implications of these findings for the ontogenesis of thymus-derived lymphocytes, and for the possible traffic of Thc within and from the thymus, are discussed.

Synergistic and suppressive interactions among mouse T lymphocytes in the response to phytohemagglutinin.

A synergistic effect in the proliferative response to phytohemagglutinin (PHA) can be observed in cultures containing a mixture of mouse CBA/Ca lymph node cells (LNC) and syngeneic CBA/T6T6 thymocytes (ThC) when compared to cultures containing only one cell type. This effect was analyzed, at various days of culture and in LNC-ThC mixtures of different ratios, by comparing the origin of the cells in mitosis (detected by caryotypic analysis), the stimulation of DNA synthesis, the number of blasts, and the percentage of blasts labeled after pulses of [3H]thymidine (detected by autoradiography). The following conclusions were reached: (a) ThC are induced to proliferate by the presence of LNC, while they are almost unresponsive to PHA when cultured alone; and (b) the strongest "synergistic" effect is exerted on LNC, whose proliferation is markedly enhanced. Evidence is presented that this last effect is not specific to the presence of ThC, but results from a dilution of LNC which retards the time when the culture reaches a critical concentration of blasts, above which proliferation progressively stops. Thus, conditions of culture allowing the response to PHA of a low concentration of LNC leads to the most prolonged T-cell proliferation. These observations may be relevant to the types of T-cell interactions, "synergistic" or "suppressive," occurring during in vitro or in vivo immune responses.

Study of the cells proliferating in parent versus F hybrid mixed lymphocyte culture.

Caryotypic analysis of the cells dividing in mouse parent-hybrid MLC showed an F1 hybrid cell proliferation, which varied depending upon the source of lymphoid cells used: strong in spleen MLC (sometimes equal to that of the parental cells), less marked in lymph node cell MLC, and most often absent in MLC between cortisone-resistant (CR) thymocytes. MLC between parental spleen cells and F1 CR thymocytes showed, however, that in certain conditions of culture F thymocytes can also proliferate. Using parental or F1 spleen cells lacking T lymphocytes, it was found that F1 cell proliferation is entirely dependent upon the presence of parental T cells, but does not require the presence of T lymphocytes among the F1 cells. Immunofluorescence analysis of the blasts observed in one-way MLC showed that about 70% of the parental blasts were T blasts, and 25%B blasts (containing a high proportion of plasmablasts); among the F1 blasts, there was also the same percentage of B blasts and plasmablasts, but many of the T blasts bore only small amounts of T-cell antigen (MTLA), and there was also about 20%of unstained blasts, possibly T blasts bearing MTLA in amounts undetectable by immunofluorescence. The possibility is discussed that the F1 responding T cells belong to a subpopulation performing a suppressive function; MLC lacking F1 T cells showed increased [3H] thymidine incorporation. The proliferation and differentiation of parental and F1 B cells may result mainly from an unspecific, "polyclonal" triggering.

Interleukin 3 perfusion in W/Wv mice allows the development of macroscopic hematopoietic spleen colonies and restores cutaneous mast cell number.

The genetically anemic W/Wv mice are characterized by the inability of their bone marrow cells to form macroscopic pluripotent hematopoietic colonies in the spleen of irradiated recipients upon transfer (colony-forming units). Furthermore, they almost totally lack mast cells, notably in the skin. In the present study, we have tested the effect of recombinant murine interleukin 3 (rmIL-3) on W/Wv mice hematopoiesis. Transfer of W/Wv bone marrow cells into lethally irradiated recipients perfused with rmIL-3 is followed by the appearance of macroscopic spleen colonies. Moreover, perfusion of rmIL-3 in W/Wv mice: (a) restores almost normal total numbers of hematopoietic precursors (colony-forming cells), but without modification of anemia; and (b) leads to the appearance of a normal number of mastocytes in the skin.

The mouse gut T lymphocyte, a novel type of T cell. Nature, origin, and traffic in mice in normal and graft-versus-host conditions.

Lymphocytes of the mouse intestinal mucosa, identified in tissue sections or purified suspensions of intraepithelial lymphocytes as T cells (gut T lymphocytes [GTL]), were studied in normal mice or in beige mice (the equivalent of the Chediak-Higashi syndrome in man, characterized by giant granules in various cell types, including mast cells). Mice were studied in normal or in germ-free conditions, or during a graft versus host (GVH) reaction resulting from the injection of parental thymocytes into lethally irradiated F1 mice, a condition leading to massive accumulation of T lymphocytes of donor origin in the host gut mucosa. In normal as well as in GVH conditions, a high percentage of the gut IE lymphocytes contain granules (up to 80% in the beige mouse). These granules have ultrastructural, hostochemical and other features resembling those of mast cell granules; in beige mice, up to 50% of them can be shown to contain histamine. Granulated T cells are also found in the lamina propria. It appears that the GTL may progressively lose their surface T antigens when the granules become more developed. Kinetics of [3H]TdR labeling of the GTL, transfer experiments with T cells of various origins, selective [3H]TdR labeling and selective irradiation of the Peyer's patches (PP), and effect of thoraic duct (TD) drainage led to the conclusion that GTL are the progeny of T cells stimulated to divide in the PP microenvironment, which endows them with a gut-homing tendency. From the PP, these cells follow a cycle, migrating to the TD and to the blood to colonize the whole intestinal mucosa, the majority of them as dividing cells undergoing a single round of traffic, with some probably able to recirculate and becoming a more long-lived variety. Antigenic stimulation within the PP is necessary for the emergence of GTL progenitors, but their gut-homing property is unrelated to the antigen as shown with fetal gut grafts, notably in GVH where grafts syngeneic to the host or donor become similarly infiltrated by GTL. On the basis of their properties and of further evidence to be reported elsewhere, it is proposed that GTL belong to a special class of T lymphocytes, related to the immune defenses of the mucosal systems in general, and capable of acting as progenitors of mucosal mast cells.

Redirection of tumor metastasis by expression of E-selectin in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Cytotoxic differentiation of mouse gut thymodependent and independent intraepithelial T lymphocytes is induced locally. Correlation between functional assays, presence of perforin and granzyme transcripts, and cytoplasmic granules.

Mouse gut intraepithelial lymphocytes (IEL), whether thymodependent (CD4+ or CD8 alpha/beta +; TCR-alpha/beta +) or thymoindependent (CD8 alpha/alpha +; TCR-alpha/beta + or -gamma/delta +), all display cytotoxic activity in a "redirected lysis assay" using anti-CD3 or anti-TCR beta or delta chains secreting hybridomas as targets; this is also observed with IEL of germ-free mice, indicating that this activity, which is absent in peripheral T lymphocytes, does not require stimulation by bacterial antigens. Perforin and granzyme transcripts are detectable in unselected gut IEL, in contrast to normal T lymphocytes of peripheral lymphoid organs. Cytological labeling (with [3H]DFP) of IEL smears reveals labeled granules (i.e., containing serine-esterases, presumably granzymes) in all subsets of gut IEL. This indicates that the gut micro-environment has an inductive role on the cytotoxic differentiation of lymphocytes of various origins when they reach the gut wall to become IEL.

Tumor necrosis factor is a critical mediator in hapten induced irritant and contact hypersensitivity reactions.

We examined the role of cytokines in the cutaneous response to the application of trinitrochlorobenzene (TNCB) in both nonsensitized and sensitized mice, i.e., in the irritant reaction (IR) and contact hypersensitivity reactions (CH). When administered immediately before challenge, anti-tumor necrosis factor (TNF) antibody abrogated the ear swelling response in CH; antibody directed against interferon gamma or antibodies to both granulocyte/macrophage colony-stimulating factor and interleukin 3 (IL-3) had a partial inhibitory effect; anti-IL-2 receptor antibody had no effect. Anti-TNF prevented the various features of the CH, as seen on histological sections, e.g., leukocyte infiltration and hemorrhages within the dermis and keratinocytes necrosis. Anti-TNF antibody also prevented the IR. The presence of TNF mRNA was evaluated on Northern blots; TNF-alpha mRNA was detectable in an untreated ear, increased after the application of TNCB in nonsensitized mice, and was highest in sensitized mice. TNF mRNA accumulation, which was evident 0.5 h after hapten application and lasted greater than 72 h, was abolished by treatment with anti-TNF antibody, thus suggesting an auto-amplification of TNF production. The cellular origin of TNF mRNA was explored by in situ hybridization; basal keratinocytes showed the highest labeling, but TNF mRNA was also detectable in cells of the dermal infiltrate. After hapten (TNCB) application at sites susceptible (the ear) or resistant (the foot pad) to CH or IR, a close correlation was observed between TNF mRNA accumulation and the intensity of the inflammatory reaction. The major role played by TNF in both the CH and the IR explains the histologically similar aspects of these reactions and the extreme variability of these reactions at various anatomical sites.

Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease.

Lethally irradiated mice were injected with semiallogeneic, T-depleted bone marrow cells and an amount of peripheral T lymphocytes sufficient to induce graft-vs.-host disease (GVHD) becoming apparent on the second week after the graft and leading to an increasing mortality rate within the following weeks (greater than 90% mortality within 80 d). Mice receiving bone marrow cells alone had no GVHD and were used as controls. Beginning on day 8, mice with GVHD were injected weekly with 2 mg of either rabbit anti-mouse recombinant tumor necrosis factor/cachectin (TNF-alpha) IgG, or normal rabbit IgG. On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD. The anti-TNF treatment resulted in an almost complete prevention of the severe lesions seen in the mice treated with normal rabbit IgG, i.e., the skin epidermal cell necrosis, foci of lichenoid hyperplastic reactions, and loss of the hypodermic fat; in the gut dilatation with marked flattening of the villi and elevation of the crypts, with increased numbers of mitoses and isolated crypt cell necrosis. In addition to preventing these acute lesions, anti-TNF treatment resulted in a significantly decreased mortality (approximately 70% survival at 80 d). These results suggest that during acute GVHD, the activation of grafted lymphocytes leads to a local release of TNF in the cutaneous and intestinal mucosae, which induces epithelial cell alterations and increases the inflammatory reaction.

Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death.

Mice injected with anti-Fas antibody die within a few hours with total liver destruction due to massive apoptosis of hepatocytes. We show that this is preceded and accompanied by the sequential activation of cysteine proteases of the interleukin 1 beta-converting enzyme (ICE) and CPP32 types in the cytosol of the hepatocytes, and that proCPP32 cleavage and enzymatic activity can be prevented by intravenous injections of the tripeptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), an inhibitor of ICE-like proteases. Four Z-VAD.fmk injections at 1-hour intervals abolished all signs of liver damage after anti-Fas antibody injection and resulted in 100% long-range recovery, without residual tissue damage, from a condition otherwise uniformly fatal within < 3 hours. This treatment was effective even when delayed until some liver DNA degradation was already detectable. Injections of the tetrapeptide Ac-YVAD.cmk, more specific for the ICE-like subfamily of cysteine proteases but less cell permeable, also gave protection, but at higher doses and when injections started before that of anti-Fas antibody. These observations afford a way of temporarily modulating a number of apoptotic processes in vivo and may have important therapeutic implications in some human diseases.

Oligoclonal repertoire of the CD8 alpha alpha and the CD8 alpha beta TCR-alpha/beta murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells.

The epithelium of the small intestine in normal euthymic mice contains a large number of intraepithelial lymphocytes (IEL), some of which bear a T cell receptor alpha/beta (TCR-alpha/beta). About half of these TCR-alpha/beta IEL display the CD8 alpha alpha phenotype and the remaining have the CD8 alpha beta or the CD4 phenotypes. To examine whether TCR-alpha/beta IEL have a TCR-beta chain repertoire as diverse as that of TCR-alpha/beta lymph node lymphocytes (LNL), we used a recently described PCR technique that allows a global analysis of the TCR-beta chain repertoire. Within any given mouse, the repertoires expressed in both CD8 alpha alpha and CD8 alpha beta TCR-alpha/beta IEL populations are oligoclonal and nonoverlapping between the two subsets. The clones are largely conserved through the length of the small intestine of the same individual. However, genetically identical individuals raised under indistinguishable environmental conditions display distinct oligoclonal repertoires. Those findings indicate that few cells of CD8 alpha alpha or of the CD8 alpha beta phenotype are responsible for the repopulation of the intestinal epithelium.

Post-thymic T lymphocyte maturation during ontogenesis.

Peripheral T lymphocytes from newborn (4-6-d-old) mice, isolated from the spleen or lymph nodes, show phenotypic features of immature cortical thymocytes, such as high frequencies of proliferating cells and of peanut lectin-binding cells. These are features of peripheral T cells of recent thymic origin, as shown by in situ labeling of thymocytes and subsequent observation of the migrants to the spleen, which were mainly peanut lectin-binding cells. The function of newborn peripheral T cells was compared, on a per T cell basis, with that of thymocytes and of fully mature peripheral T cells of the adult, using preparations of newborn lymph node cells containing approximately 80% of T lymphocytes. They were strikingly (about 10-fold) less competent than adult T cells in their phytohemagglutinin responsiveness, their capacities to induce a graft vs. host reaction, to proliferate in the mixed lymphocyte reaction, and to help B lymphocytes in a humoral response in vivo and in vitro. In contrast, newborn T lymphocytes were comparable to those of adults in their capacity to generate cytotoxic T lymphocytes. No suppressive effect of newborn T lymphocytes could be demonstrated in several of these assays. These results argue for an asynchronous maturation of two T cell subsets during ontogeny and demonstrate that at least some T lymphocytes leave the thymus as immature T cells resembling cortical thymocytes and further mature at the periphery. Investigation of mice submitted to thymectomy of 5 d of age showed that these incompetent post-thymic T lymphocytes are capable of considerable expansion and maturation in the peripheral lymphoid organs in the absence of a thymic influence.

Gut mucosal mast cells. Origin, traffic, and differentiation.

Gut mucosal mast cells (MMC), which are nearly absent in normal mice are abundant during nematode infection. In normal mice, study of MMC precursors (MMC-P: cells giving rise to MMC colonies in the presence of IL-3) show that: (a) their frequency, judged by limiting dilution is very high in bone marrow (BM) and gut, and very low in most lymphoid organs and thoracic duct lymph (TDL); (b) gut MMC-P are Thy-1- Lyt-1-2- and are not rapidly replicating; (c) they are the progeny of less differentiated BM MMC-P which are attracted from the blood to the gut mucosa by local factor(s), other than antigen and T cell factors (since normal amounts of gut MMC-P are found in germ-free, nude, and newborn mice). In mice bearing the Wehi 3 tumor (which releases enough IL-3 to produce detectable blood levels) spleen and mesenteric lymph nodes (LN) show increased MMC-P frequency, the greatest increase being in the gut and BM, where numerous differentiated MMC are found. In Nippostrongylus brasiliensis (Nb)-infested mice (known to develop a large, T cell-dependent, gut MMC infiltration), gut MMC-P proliferation is induced by IL-3 released from gut mucosal Thy-1+ Lyt-2- cells, whose in vitro IL-3 release capability is much higher than that of similar cells from normal mice. Both Nb-stimulated T blasts and proliferating MMC-P undergo cyclic traffic, migrating into the TDL and then seeding the whole length of the gut (a process which allows a widespread immune defense after a local antigenic stimulus). Experiments using 2-d interruption of this traffic and fetal gut grafts, suggest that the continuous homing of T blasts back to the gut which leads to permanent Nb-stimulated IL-3 release, is essential for the full maturation of MMC. Transfer experiments in the rat show that TDL circulating MMC-P rapidly mature into MMC when they home back to the Nb-infested gut. It is proposed that gut MMC arise after several stages of progressive differentiation of MMC-P, influenced both by IL-3 and unidentified gut factor(s).

Interleukin 3 perfusion prevents death due to acute anemia induced by monoclonal antierythrocyte autoantibody.

We have evaluated the therapeutic activity of rIL-3, in comparison with recombinant granulocyte-macrophage CSF (rGM-CSF) and recombinant erythropoietin (rEpo), on a lethal form of acute anemia induced by a single injection of a monoclonal IgG1 anti-mouse RBC (MRBC) autoantibody. Continuous perfusion of rIL-3 before the administration of anti-MRBC mAb prevented animals from the death due to anemia with a rapid recovery in greater than 90% of the cases, while only partial protection (one third of the cases) was obtained by rEpo perfusion, and no protection by rGM-CSF. Since the anti-MRBC mAb induced a marked agglutination of RBC in spleens and livers, and subsequent hemodynamic failure may be an additional contributing factor to the animals' death, the activation of Fc gamma receptor-dependent phagocytosis by rIL-3, as well as the increased number of monocytes/macrophages resulting from rIL-3 perfusion, may also facilitate rapid elimination of these agglutinated RBC, resulting in the further amelioration of the animals' survival. Our results suggest that the therapeutic effect of rIL-3 on anti-MRBC autoantibody-induced anemia is achieved by: (a) its activity to promote the growth and differentiation of erythroid progenitors responsive to Epo and of monocyte/macrophage lineage; and (b) its activity to enhance the phagocytic activity of macrophages to efficiently eliminate agglutinated RBC in spleens and livers.