The development of lymphocytes with T- or B-membrane determinants in the human foetus

The development of T- or B-membrane determinants on human foetal lymphoid cells was studied by the direct immunofluorescence technique, using a tetramethyl rhodamine isothiocyanate (TRITC) labelled horse antihuman T-cell conjugate (ATC) for the detection of T lymphocytes and a fluorescein isothiocyanate (FITC) labelled goat antihuman Fab conjugate for the demonstration of Ig-bearing B lymphocytes. Human foetal lymphocytes were also tested for spontaneous rosette formation with sheep red blood cells (SRBC).

Cell suspensions of liver, spleen, thymus, bone marrow and blood of twenty-five human foetuses of 5·5–26 weeks of gestational age have been investigated. ATC-positive lymphoid cells were first seen in the liver at 5·5 weeks; E rosette-forming cells (ERFC) and Ig-bearing lymphoid cells were first found at 9 weeks. ERFC were also present in the thymus at 9 weeks. By 12 weeks, fluorescent B and T lymphocytes were found in bone marrow and spleen. ERFC were also found in bone marrow at this age, but not in spleen. At 15 weeks, more than 80% of blood lymphoid cells had T or B determinants.

A difference in the reactivity of lymphoid cells with the ATC and their capacity to form E rosettes was observed. In liver and spleen, the ATC determinant was detectable before the SRBC receptor. In bone marrow, blood and thymus the ATC determinant was found on a higher percentage of lymphoid cells than was the SRBC receptor when those organs were first investigated. During the entire investigated period of gestation, the majority of lymphoid cells in liver and bone marrow did not react with either of the conjugates, nor did they form E rosettes. In all organs investigated, except in the thymus, lymphoid cells were occasionally seen which reacted with both conjugates. By the 16th week of foetal age, more than 90% of lymphoid cells in thymus, spleen and blood had acquired T- or B-membrane determinants.

     

Studies on the differentiation of B lymphocytes in the mouse

The capacity of CBA mouse B lymphocytes to bind polyvalent rabbit anti-mouse immunoglobulin antibody, even when this reagent is present in low concentration, was used as an index of a lymphocyte's B cell status. Various mouse tissues were held for 30 minutes at 0° with 0.2 μ/ml of ¹²⁵I-labelled anti-Ig, and smear preparations of washed cells were examined, following radioautography, for their content of B cells. The survey covered two areas of B cell differentiation, namely the spontaneous emergence of B cells in the mouse foetus, and the development of B cells in lethally irradiated mice that had received early foetal liver as a source of haematogenous stem cells.

The foetal survey suggested a multifocal origin of B cells, commencing 3 days before birth, in all major sites of erythromyelopoiesis, namely liver, spleen and bone marrow. Lymph nodes showed B cells later than did these organs, and thymus contained virtually no B cells at any stage. A rapid influx of B cells into lymph nodes took place shortly after birth. The CBA mouse is born with somewhat over 10⁵ B lymphocytes in toto, of which the majority are in liver, spleen and blood.

The irradiation-recovery study showed: (1) that B cells are, statistically, more sensitive to high dose irradiation than T cells; (2) that a B cell-free stem cell source can repopulate the B cell pool; (3) that repopulation is surprisingly slow; and (4) that numerical and functional recovery of B cells parallel each other to a reasonable degree.

The significance of the results is discussed from the viewpoints of lymphocyte differentiation and immunological tolerance.

     

Effect of thymus factor on human precursor T lymphocytes

A pig thymus factor is shown to transform in vitro human immature lymphoid cells (from cord blood, bone marrow and foetal tissue) into mature T lymphocytes forming spontaneous rosettes with sheep erythrocytes. No effect was observed on normal mature lymphocytes (T or B cells), suggesting a selective activity of the thymus factor for precursor T lymphocytes.

When lymphocytes from various patients were treated with the thymus factor, the most pronounced effect was found on cells from T cell-deficient patients. This finding suggests that these patients were lacking such a humoral thymus factor and were not genuinely deficient in their lymphocytes. Our results indicate a potential diagnostic value of the thymus factor, especially in the analysis of immunodeficiency diseases.

     

Immunoglobulin-bearing lymphocytes of the chicken. I. Heavy chain immunoglobulin commitment and organ distribution

When purified anti-immunoglobulin light chain antibodies were used in indirect immunofluorescence or labeled with 125I for autoradiographic staining, a similar percentage of Ig-bearing lymphocytes were detected by both techniques in lymphoid cell suspensions from the thymus or blood of 8-14-week-old chickens. However, a larger proportion of Ig positive lymphocytes were detected in suspensions of bursal cells by the more sensitive autoradiographic method, suggesting a lower surface density of Ig: perhaps on newly differentiated stem cells. In thymus and spleen suspensions, the proportions of Ig positive lymphocytes carrying mu and gamma-chains were roughly equal, whereas in the B cell populations of the bursa and blood, cells carrying surface gamma-chains predominated. IgA-bearing lymphocytes were only a minor population (< 5%) in lymphocyte suspensions prepared from the thymus, bursa, blood and spleen of adult chickens, but formed almost 50% of the Ig-bearing lymphocytes in the caecal tonsils.     

Partial reconstitution of V(D)J rearrangement and lymphocyte development in RAG-deficient mice expressing inducible, tetracycline-regulated RAG transgenes

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.     

Free immunoglobulin light chain synthesis by human foetal liver and cord blood lymphocytes.

Immunoglobulin (Ig) synthesis by immature B lymphocytes from human foetal liver and cord blood has been investigated. Seven out of fifteen preparations of foetal liver cells and eight out of eleven cord bloods synthesized Ig detectable by biosynthetic labelling. All cultures of foetal lymphocytes with detectable Ig synthesis secreted free light chain. Two of these cases also synthesized free mu heavy chain. Cord blood lymphocyte synthesis patterns were variable ranging from free light chain as the only secreted Ig product to balanced synthesis of heavy and light chains. No cord blood cultures synthesized detectable free mu chains. Free Ig-light-chain synthesis appears to be associated with normal immature B lymphocytes and the results are discussed in relation to the B-cell differentiation pathway.     

Demonstration of immunoglobulin on the surface of thymus lymphocytes

Immunoglobulin molecules on the surface of mouse thymus cells have been shown by immunofluorescence. Ninety-five to 100 per cent of thymus lymphocytes were found to bind polyvalent rabbit anti-mouse immunoglobulin, although the density of the fluorescence was much less than that on the surface of B lymphocytes derived from the spleen.

Two purified antisera, an anti-IgM and an anti-kappa chain serum, also stained the cells.

The resynthesis of these immunoglobulin molecules in vitro was demonstrated after they had been removed with pronase.

     

Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs

Adult, male Lewis rats received a single injection of 5-bromo-2'-deoxyuridine (BrdUrd) i.v. to label proliferating cells in the S phase of the cell cycle. After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed. In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying kappa light chain, CD5+, CD4+ and CD8+ cells, respectively. On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody. B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches. Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold. Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd-labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%). One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes. Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h. Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.     

Lymphocyte markers in the bovine foetus

The ontogeny of lymphocyte surface markers was studied in thymus, retropharyngeal lymph nodes, spleen, bone marrow, and liver of 48 bovine foetuses, 80–280 days of gestation. The markers examined were the spontaneous (E) rosette against sheep erythrocytes (SRBCs) in foetal calf serum (E/FCS), in medium supplemented with gelatin (E/CFG), and in medium supplemented with dextran (E/dextran), the erythrocyte-antibody (EA) rosette for immunoglobulin (Fc) receptors, the erythrocyte-antibody-complement (EAC) rosette for complement receptors, and the immunofluorescence test (IFT) for surface-membrane immunoglobulins (SmIg). E-rosette-forming cells (RFCs) occurred in the thymus in substantial numbers throughout the period studied, in spleen and lymph nodes only after 160 days, and in bone marrow and liver rarely. The dynamics of E rosettes formed in the three supplements suggested that each supplement might be supporting rosette formation by different populations of cells, albeit considerable overlap might be expected. Organ distribution and development of EA and EAC RFCs did not parallel the expected ontogeny of any class of lymphocytes. Cells carrying SmIg developed mainly in spleen and lymph nodes. Towards the end of gestation some cells in liver also had SmIg.     

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.     

Phylogeny of lymphocyte heterogeneity. I. Membrane immunoglobulins of teleost lymphocytes.

Immunofluorescence studies of bream lymphoid tissues revealed that our 90% of the lymphocytes from blood, anterior kidney, spleen and thymus exhibited membrane immunoglobulin determinants. Furthermore, a majority of such cells were observed to undergo patching and capping when the membrane proteins were complexed with antisera to fish serum immunoglobulins. Lactoperoxidase catalysed radioiodination, detergent lysis and immunoprecipitation with appropriate antisera were employed to study the properties of this membrane immunoglobulin. Quantification, by inhibition of immunoprecipitation with serum immunoglobulin, indicated the average amount of immunoglobulin determinants for bream lymphocytes from either blood, anterior kidney, spleen or thymus to be about half that present on mouse B cells. Physiochemical characterization of labeled membrane immunoglobulin from bream lymphocytes suggested that only one class of immunoglobulin heavy chain was present and that about one-half of this material resembled the monomeric (2H-2L chain) IgM-like proteins present in bream serum. A major unanswered question raised by this study was whether or not certain of the bream membrane immunoglobulin determinants were associated with molecules that did not resemble "classical" immunoglobulins.     

Differential regulation of V(D)J recombination during development of avian B and T cells

The lymphold immune system is comprised of two major cell types,B cells and T cells, originally identified in avian species.Although both lineages arise from hematopoletic stem cells,avian B cells require a period of development in the bursa ofFabricius while T cells undergo development in the thymus. Eachcell type expresses a lineage-specific antigen receptor encodedby genes created by the rearrangement of Individual membersof variable (V), diversity (D), and joining (J) gene segmentfamilies during embryonic development. In this report, we demonstratethat productive rearrangement of the TCR ß gene occursexciusively in the thymus during normal development. TCR ßrearrangements involving gene segments from the V_ß1gene family can be detected beginning on day 12 of development,while rearrangements involving the other family of V_ßgene segments, V_ß2, were first detected on day 14of embryogenesis. In contrast, productive rearrangements ofIg light (IgL) and heavy (IgH) chain genes were not restrictedto the bursa of Fabricius. Instead, V_H-DJ_H heavy chain rearrangementsand V_L-J_L light chain rearrangements were detected primarilyin the embryonic spleen, beginning as early as embryonic day10, even in birds bursectomized at 60 h of development. Withinthe spleen, Ig rearrangementwas confined to the subset of cellsthat express the chB6 surface protein. Unlike bursal lymphocytes,which express the recomblnase activating gene (RAG)-2 but notRAG-1, splenic B cell precursors also express RAG-1. The dataindicate that, while B cell precursors initiate recombinationprior to migration of the bursa of Fabricius, T cell precursorsundergo V(D)J recombination following migration to the thymus.Thus, distinct developmental mechanisms appear to regulate theprocess of receptor rearrangement during avian B and T celldevelopment.     

Immunofluorescent detection of surface antigens specific to T and B lymphocytes in the chicken

Rabbit antisera specific for chicken T and B cells as judged by surface immunofluorescent staining have been raised. Specificity was established by the staining of thymus and bursal cell suspension and by the effects of thymectomy and bursectomy on the staining of peripheralized lymphocytes. Furthermore, double labeling experiments showed that anti-T and anti-B sera reacted with different populations of blood lymphocytes. Comparable numbers of cells in blood and spleen stained for B and light chain determinants. No evidence for “null cells” was obtained. There was little change in the percentage of cells staining in the various lymphoid organs from 4 days to 12 months of age. The thymus contained approximately 7 % B cells, although no T cells were demonstrable in the bursa. One antiserum showed only thymocyte specific antibodies not reacting with peripheral T cells. The specific B and T markers seem to be acquired during differentiation within the appropriate central lymphoid organ. Demonstrable surface immunoglobulins appear later in ontogeny than the B antigens. The majority of cells bearing the B marker in bone marrow were large cells lacking surface light chain determinants.     

Increase of CD5(+) B cells during adolescence in female mice

We examined physiological changes in CD5(+) B lymphocytes in mice associated with aging (from 1 or 5 to 50 weeks of age). The most predominant populations of lymphocytes among these mice were mainly CD5(-) B cells in the spleen and peritoneal cavity, and T cells in the thymus. However, in the spleen, CD5(+) B cells increased from 1 to 15 weeks, and then decreased with aging. In the thymus, CD5(+) B cells increased from 3 to 9 weeks of age, and subsequently became more predominant than CD5(-) B cells. In the peritoneal cavity, CD5(+) B cells increased from 5 to 9 weeks of age, became the most predominant population in lymphocytes at 7 to 9 weeks of age, and decreased with aging. The proportion of CD5(+) B cells in total B cells increased from 5 to 7 or 9 weeks of age, and then decreased with aging, with the highest proportion at 9 weeks of age in the spleen (15%), thymus (94%), and peritoneal cavity (54%). These findings indicate that CD5(+) B cells increase physiologically during mouse adolescence, and subsequently decrease with aging.     

Bovine lymphocytes: recognition of cells forming spontaneous (E) rosettes.

About 20% of thymus lymphocytes from neonatal calves formed spontaneous (E) rosettes with SRBCs in medium consisting of 50-100% foetal calf serum (FCS); other media were less satisfactory. FCS was necessary both to allow rosette formation to occur and to maintain stability of the rosettes once formed. Rosettes were stable at 0 degrees C but unstable at 18 degrees C and 37 degrees C. Dead thymus cell (sodium azide treated) did not form rosettes. Treatment of thymus cells with antiserum to bovine Ig-inhibited rosette formation, but this inhibition was considered non-specific since it also occurred with normal rabbit serum. Treatment of SRBCs with neuraminidase slightly enhanced rosette formation by thymus cells, but did not induce peripheral blood lymphocytes to form rosettes. Rosette formation did not occur under a variety of conditions with normal or neuraminidase-treated human, horse, pig, rabbit, guinea-pig, chicken or autologous RBCs. SRBC rosette forming cells were also found in lymph nodes (2-14%) and spleen (less than 5%), but rarely or never in peripheral blood and bone marrow of calves and adults. In foetuses at 80 days of gestation, 49% of thymus cells formed E rosettes. Foetal lymph node cells formed E rosettes at 160 days and spleen at 180 days. Cells with membrane-bound Ig were observed by IFT; their distribution did not coincide with the occurrence of E rosettes. E-rosette formation might be a marker for a subpopulation of bovine T cells.     

Repopulation of lymph nodes and spleen in thymus chimeras after lethal irradiation and bone marrow transplantation: dependence on the age of the thymus

CAP and Lewis rats were thymectomized and received a syngeneic thymus graft followed by lethal irradiation and syngeneic bone marrow transplantation. In three groups (A: recipient 15 months old, thymus graft 3 months old; B: recipient 3 months old, thymus graft 15 months old; C: recipient and thymus graft both 3 months old), we performed an immunohistologic analysis of the splenic white and red pulp and the paracortical zone of the lymph nodes. The repopulation of these regions was demonstrated with monoclonal antibodies that react with Thy-1 positive cells, peripheral T cells, T helper cells, and T non-helper cells. In the splenic red pulp, more Thy-1 positive lymphocytes were found in group B than in group C. The proportion of T lymphocytes and T helper lymphocytes in the region of the periarteriolar lymphocyte sheath of the splenic white pulp was higher when a young thymus was transplanted (groups A and C) than when an old one was (group B). In contrast, in the splenic red pulp, more T lymphocytes were found in group A than in groups B and C. In the paracortical zone of the lymph nodes, this was demonstrable only for group C versus group B. The proportion of T non-helper lymphocytes in the region of the splenic red pulp was higher in group B than in group C. These results indicate that the repopulation of lymph nodes and spleen after transplantation of an old thymus is delayed, quantitatively reduced, and qualitatively different (more T non-helper lymphocytes).     

Ontogeny of the immune system of the brushtail possum, Trichosurus vulpecula

The numbers and distribution of T and B cells in the thoracic thymus, spleen and intestinal tissue and the proliferation of T lymphocytes were examined during pouch life and in the adult to determine when the developing brushtail possum reaches immunological maturity. CD3-positive cells were observed in the thoracic thymus at day 2 post-partum indicating that the thymus produces T lymphocytes at or soon after birth. By day 25 the thymus was fully populated with CD3-positive T lymphocytes and they were observed in distinct regions of the cortex and medulla. By day 48 post-partum, B and T lymphocytes were identified in the follicles and parafollicular areas of the spleen. Although the numbers of T and B cells in the spleen increased significantly from day 25 to day 100 post-partum (P < 0.005), fewer cells were present at day 150 post-partum than in the adult (P < 0.05). Peyer's patches were not observed in the intestines up to day 73 post-partum. However, both T and B cells were observed in the intestinal lymph nodes. Although the T lymphocytes at weaning showed a proliferative response, the response was not as great as that observed in the adult possum. Thus, the immune system of the possum is not fully developed at weaning but continues its development after pouch life.     

Ontogeny and development of extrathymic T cells in mouse liver.

We previously demonstrated that the liver may be a major site of extrathymic T-cell differentiation in mice. In the present study, the ontogeny and subsequent development of such T cells in the liver and other organs were investigated. This study was possible because these T cells have T-cell receptors (TcR) of intermediate intensity (i.e. intermediate TcR cells) and constitutively express a high level of interleukin-2 receptor beta chain (IL-2R beta). Therefore the two-colour staining for CD3 (or alpha beta TcR) and IL-2R beta identifies even a small proportion of intermediate TcR cells. The total numbers of mononuclear cells obtained from the liver, thymus and spleen varied from foetal to adult life. Especially in the liver, many haematopoietic cells were present in the parenchymal space at the foetal stage. There were no lymphocytes in the sinusoidal lumen at this period. In contrast, lymphocytes appeared in the hepatic sinusoids after birth and increased with ageing. Phenotypic analysis revealed that intermediate TcR cells appeared in the liver and spleen on Day 4 after birth. Bright TcR cells of thymic origin were also present in the peripheral organs on Day 4. Thereafter, intermediate TcR cells increased in the liver, whereas bright TcR cells increased in the periphery as a function of age. Similarly, thymectomized and congenitally athymic mice had mainly intermediate TcR cells in the liver and, to some extent, periphery. It is concluded that intermediate TcR cells, possibly of extrathymic origin, are generated only after birth and expand with ageing.     

Reactivity differences among human T cells from blood and lymphoid organs, analysed by limiting dilution: correlation with specific gravity and binding of peanut lectin.

Human T cells from peripheral blood, cord blood, thymus, spleen and lymph node were analysed for their proliferative response capacity to mitogens, for their specific gravity and size, and for their binding capacity of peanut agglutinin. A close correlation was found between these parameters: thymocytes and T cells from spleen were consistently heavier and smaller, and showed a lower proliferative response capacity, than T cells from blood or lymph node. Similarly, within each cell population, the small, heavy cells were least reactive. The limiting-dilution analysis revealed that heavy T cells from peripheral blood contain a lower number of reactive cells than the light peripheral T lymphocytes. Because heavy T cells from the thymus bound more peanut lectin than did light cells, it is speculated that the differences between T cells of high and low specific gravity might reflect differences in maturation level.