Lymphocytes In Adult Mice Research Articles

HSC-independent definitive hematopoiesis persists into adult life

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Distinct binding patterns of fucose-specific lectins from Biomphalaria alexandrina and Lotus tetragonolobus to murine lymphocyte subsets

The putative expression of distinct terminally fucosylated glycoconjugates among murine lymphocyte subpopulations was sought using a Biomphalaria alexandrina-derived lectin (BaSI), of proven specificity to a fucosyllactose determinant, and the fucose-binding lectin from Lotus tetragonolobus seeds. Direct labeling of isolated lymphocyte subsets in suspension as well as immuno-histochemical and two-dimensional Western blotting assays demonstrated the exclusive expression of the BaSI-reactive ligand among multiple isoforms of two major 95 and 92 kDa and a minor 82 kDa acidic glycoproteins, selectively localized to the splenic marginal zone B lymphocytes of adult mice. The expression of the L. tetragonolobus lectin-reactive ligand was, on the other hand, restricted primarily to a single homogeneous 50 kDa acidic glycoprotein associated with a subset of the mature (PNA −) medullary thymocytes of adult mice as well as a minority of the immature (PNA +) thymocytes within the deep cortical region in newly born mice. The significance of these findings is discussed in relation to mechanisms that govern lymphocyte development and homing.

Monoallelic Expression of Reactivated Imprinted Genes in Embryonal Carcinoma Cell Hybrids

Though DNA methylation is necessary to maintain monoallelic expression of imprinted genes, it is still unclear whether it represents the primary mark. Here we ask whether the imprinting mark is still present in terminally differentiated somatic cells in which the transcription of embryo-specific imprinted genes was shut off. For such analysis H19 and Igf2 genes were activated by inducing differentiation of (mouse embryonal carcinoma cell × mouse lymphocyte) hybrid cell clones. Although lymphocytes do not express H19 and Igf2, both genes are reactivated in a proper monoallelic manner in hybrid cells. Analysis of the upstream region of the H19 gene confirmed maintenance of differential methylation of the active and inactive H19 genes of lymphocyte origin, although a tendency toward in vitro induced hypermethylation was apparent. We conclude that the imprints of the H19, U2af1-rs1, and Igf2 genes are maintained in lymphocytes in adult mice.

CD98: A Type II Transmembrane Glycoprotein Expressed From the Beginning of Primitive and Definitive Hematopoiesis May Play a Critical Role in the Development of Hematopoietic Cells

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.

Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice.

Lymphoid enhancer factor 1 (LEF-1) is a sequence-specific DNA-binding protein that is expressed in pre-B and T lymphocytes of adult mice, and in the neural crest, mesencephalon, tooth germs, whisker follicles, and other sites during embryogenesis. We have generated mice carrying a homozygous germ-line mutation in the LEF-1 gene that eliminates its protein expression and causes postnatal lethality. The mutant mice lack teeth, mammary glands, whiskers, and hair but show no obvious defects in lymphoid cell populations at birth. The LEF-1-deficient mice also lack the mesencephalic nucleus of the trigeminal nerve, the only neural crest-derived neuronal populations. Together, the pattern of these defects suggest an essential role for LEF-1 in the formation of several organs and structures that require inductive tissue interactions.

Developmental shift in the patterns of interleukin production in early post-natal life.

In mice, immunological tolerance to self is established in the perinatal period, when tolerance susceptibility to allogenic tissues is higher than in adults. We have now investigated whether this could result from developmental regulation of effector functions of T cells exposed to specific antigens, by studying the "natural" or T cell receptor-induced expression of several interleukin genes. We used qualitative and quantitative polymerase chain reaction methods to study interleukin (IL)-2, IL-4, IL-10 and interferon-gamma mRNA expression by splenic cells at different ages. The results show that newborn peripheral cells (up to day 7), in contrast to the T lymphocytes of adult mice, express high levels of IL-4 and interferon-gamma, and very low levels of IL-2 messenger spontaneously and upon specific T cell activation. This characteristic phenotype depends on intrinsic T cell properties, as it is not due to the newborn environment.

In vivo gene delivery. Efficient transfection of T lymphocytes in adult mice

We have demonstrated that a single intraperitoneal injection of cationic liposomes complexed to a chloramphenicol acetyltransferase (CAT) gene expression plasmid can transfect the majority of splenic Thy 1.2+ T lymphocytes, as well as significant numbers of bone marrow-derived hematopoietic cells, in adult mice. CAT activity was detected in the spleen for at least 2 weeks, and there was no evidence of treatment-related toxicity. Some degree of tissue-specific transgene expression was achieved by varying the cationic lipid used.

Deletion of donor-reactive T lymphocytes in adult mice after intrathymic inoculation with lymphoid cells.

Clonal deletion of self antigen-reactive T lymphocytes is known to be a dominant mechanism of tolerance induction in the normal immune system. This report considers whether deletion of antigen-reactive T cells is also the immunologic basis for the recently described model of transplantation tolerance that follows intrathymic inoculation with allogeneic lymphoid cells. We found that the outcome of injecting Mlsa- hosts with lymphocytes from Mlsa+ donors was depletion of V beta 6+ T cells (which are known to be reactive with the Mlsa superantigen). The process was found to be specific in that a similar reduction was not seen in an irrelevant T cell population (V beta 8+) in IT injected hosts. Deletion was observed in this model only if immunosuppression with ALS or anti-CD4 accompanied intrathymic injection. When the inoculum of allogeneic lymphocytes was administered intravenously instead of intrathymically only minimal deletion was observed. The induction of transplantation tolerance by intrathymic injection of donor lymphoid cells may prove especially efficacious since it relies on deletion of only those T cells specifically reactive to donor antigens, a process analogous to tolerance induction to self antigens.

Thymostimulin effects on lymphoid organs in Ames dwarf mice

This work was undertaken to study the effects of thymostimulin (TP-1) on the immune function in Ames dwarf mice, and to relate these effects to PRL and/or GH deficiency in these animals. Male Ames dwarf mice implanted with pituitaries from normal mice under the kidney capsule, sham-operated dwarf mice and normal immature or adult mice were injected daily for five days with TP-1. In comparison to normal animals, sham-operated dwarf mice had markedly lower body, thymus and spleen weights, as well as a lower number of lymphocytes in the spleen and in the thymus and the natural killer (NK) activity of spleen lymphocytes. Ectopic pituitary transplants produced the expected enhancement of body weight gain and increased spleen and thymus weights, which reached the values found in normal (non-dwarf) animals. The numbers of lymphocytes in the spleen and thymus were significantly increased in pituitary-grafted dwarf mice, but the grafts did not modify the cytotoxic activity of NK spleen cells, or the number of peripheral white blood cells (PWBC). In sham-operated dwarf mice, TP-1 treatment did not modify the number of cells in the spleen and thymus, or the NK activity. In pituitary-grafted dwarf mice, treatment with TP-1 induced an increase in the number of spleen lymphocytes and in the NK activity of spleen cells without affecting the weight of lymphoid organs or the number of thymic cells. Plasma prolactin (PRL) and growth hormone (GH) levels of pituitary-grafted dwarf mice were not changed after TP-1 administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of Junin virus persistence in adult athymic mice.

To determine the role of T lymphocytes in adult mice infected with Junin virus, 60-day-old athymic (nu/nu) mice and their immunocompetent (nu/+) littermates were inoculated intracerebrally with 10(3) TCD50 of the XJ strain. None of them exhibited neurologic illness during a 6-month observation period, and mortality was 3% for nu/nu and 7% for nu/+ animals. The main features in infected nu/nu mice were: high viral titers in brain, reaching a late peak (6.5 log/ml) 32 days postinoculation and persisting at least 6 months; low, late viremia appearing simultaneously with the viral peak in the central nervous system (CNS) and persisting up to 3 months after infection; absence of signs of neurologic disease or histologic lesions in brain and almost no mortality; and lack of detectable circulating antibodies either in IgM or in other immunoglobulins (Igs). Circulating anti-Junin antibodies were demonstrated in IgM and other Igs in immunocompetent mice, although no infectious virus or histologic lesions could be detected. These results show an important role for T lymphocytes in the clearance of Junin virus in the CNS, as demonstrated by viral persistence induced in adult athymic mice.

T cell repopulation from functionally restricted splenic progenitors: 10,000-fold expansion documented by using limiting dilution analyses.

Mice depleted of T cells by thymectomy, lethal irradiation, and reconstitution with Thy-1-depleted syngeneic bone marrow were given graded doses of splenic T cells to see whether post-thymic cells had the ability to regenerate immune function in these hosts. Using limiting dilution methods to estimate the number of antigen- and mitogen-responsive cells in recipients 12 to 20 wk after reconstitution, we found that new helper and cytotoxic precursor cells were produced, but attained levels only 10 to 20% of normal. Because these repopulated mice were able to produce nearly normal levels of helper and cytotoxic activity in conventional, high density cultures, despite their relative paucity of precursors, we infer that their normal function in conventional assays may reflect a balanced deficiency of effector and regulatory cell types. Surface phenotyping of the progenitor cells responsible for repopulation showed that Lyt-2- cells were required for helper cell regeneration and that Lyt-2+ cells acted as progenitors only for the cytotoxic lineage, contrary to earlier speculation that the splenic Lyt-1+ 2+ (Ly-123) pool included cells antecedent to both effector lineages. Comparison of the number of injected progenitors needed to produce repopulation with the number of new precursor cells eventually produced suggests that the relevant progenitors are able to undergo 10,000-fold expansion in 12 to 20 wk. Numerical expansion in the periphery from thymic-processed cells could well be a major source of new lymphocytes in adult mice.

SIgD-negative B cells from neonatal mice do not respond to the thymus-independent antigen TNP-BA in limiting dilution cultures.

The majority of B lymphocytes in adult mice express both IgM and IgD on their surface (sIgM and sIgD). A small percentage of sIgM+ splenic B cells lack (or express very low levels of) sIgD. These cells have been termed "mu-predominant" (mu p) B cells. In neonatal mice (5 to 12 days of age), mu p B cells account for more than 50% of the sIg+ cells. There is conflicting evidence concerning the immunocompetency of mup cells in vitro. To study this question further, splenocytes from neonatal BALB/c mice were depleted of sIgD+ B cells by a panning procedure. A portion of the nonadherent (mu p) cell population was analyzed for residual sIgD+ cells by using indirect immunofluorescence in conjunction with the fluorescence-activated cell sorter (FACS). Such cells were then tested for their responsiveness to the thymus-independent (TI) antigen, trinitrophenyl Brucella abortus (TNP-BA), by using a limiting dilution culture system. Results indicate that the depletion of sIgD+ B cells and the decrease in the precursor frequency of splenocytes responding to TNP-BA are very similar, suggesting that virtually all of the responding B cells bear sIgD.

Heterogeneity of complement receptor expression on surface immunoglobulin-bearing cells from neonatal and adult mice.

The ontogeny of acquisition of complement receptors (CR) on splenic B lymphocytes from mice of varying ages was examined. Reagents used for the identification of CR included antibody- and complement-coated erythrocytes (EAC), bacteria (BAC) and bacteria treated with complement alone (BC). When whole mouse serum used as the source of complement both EAC and BAC failed to bind to human erythrocytes (C3b receptor-negative), showed binding to Raji and Daudi cells (C3d receptor) and formed rosettes with human neutrophils (C3bi receptor). Therefore these reagents bore C3d and C3bi, but not intact C3b. Although EAc and BAC detected nearly equal percentages of CR lymphocytes in adult mice, BAC bound to a higher percentage of neonatal lymphocytes (8-12%) than did EAC (1-3%). The ability of BAC to detect EAC-negative lymphocytes among neonatal spleen lymphocytes appeared to be due to the increased sensitivity of this bacterial reagent for binding to CR. Further evidence of this enhanced sensitivity was that BAC were less inhibitable than EAC by soluble antigen-antibody-complement complexes.

Pre-B Cells: Bone Marrow Persistence in Anti-µ-Suppressed Mice, Conversion to B Lymphocytes, and Recovery after Destruction by Cyclophosphamide

Abstract Chronic treatment of mice from birth with anti-µ antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-µ-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-µ treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.

Cell Surface Immunoglobulin

Abstract The differentiation of Ig-bearing lymphocytes in adult mice was studied by monitoring the appearance of IgD relative to IgM on the surface of splenocytes obtained from lethally irradiated animals reconstituted for various periods of time with adult bone marrow cells, neonatal splenocytes, or Ig- adult splenocytes. It was found that IgM appears before IgD on differentiating lymphocytes. Furthermore, the rate of appearance of IgD during differentiation of adult cells is similar to that observed with neonatal cells.