Interdigitating cells in the lymphoid tissues of bovine fetuses and calves

Summary Electron-microscopic studies of lymphoid tissues from bovine fetuses and from calves disclosed a non-lymphoid cell type in the thymus-dependent zones of secondary lymphoid tissues and in the thymus that is distinguishable from reticulum cells, epithelial and endothelial cells, and macrophages. Based on morphological and topographical criteria, the cell is identified as the interdigitating cell. In addition, studies of the tissues of normal and virus-challenged fetuses, and of conventionally reared calves, indicated that the interdigitating cells originate from monocytoid cells, which undergo differentiation in the thymus-dependent zones during an immune response.     

Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies

Endothelial cells play an essential role in immune responses by regulating the entry of leukocytes into lymphoid tissues and sites of inflammation. As an initial approach to analyzing endothelial cell specialization in relation to such immune function, we have produced monoclonal antibodies (MAB) against mouse lymph node endothelium. Three antibodies were selected: MECA-20, recognizing the endothelium of all blood vessels in lymphoid as well as non-lymphoid organs; MECA-217, which stains the endothelium lining large elastic arteries, but among small vessels is specific for post-capillary venules within lymphoid organs and tissues exposed to exogenous antigen, such as skin and uterus; and MECA-325, an antibody that demonstrates specificity for the specialized high endothelial venules (HEV) that control lymphocyte homing into lymph nodes and Peyer's patches. MECA-325 failed to stain vessels in any non-lymphoid organs tested. Immunoperoxidase studies of HEV in lymph node frozen sections, and of isolated high endothelial cells in suspensions, demonstrated that the antigens recognized by all three antibodies are expressed at the cell surface; those defined by MECA-20 and MECA-325 are also present in the cytoplasm. To study the regulation of the antigens defined by these MAB in relation to extra-lymphoid immune reactions, we assessed their expression in induced s.c. granulomas as a model for chronic inflammation. Small vessels in the granulomas were already stained by MECA-217 in the first days of development. In contrast MECA-325 detected postcapillary venules (which frequently displayed the morphologic characteristics of HEV) only from approximately 1 wk, in parallel with the development of a persistent mononuclear cell infiltrate including numerous lymphocytes. The selective appearance of the MECA-325 antigen on vascular endothelium supporting lymphocyte traffic in both lymphoid and extra-lymphoid sites suggests that this antigen may play an important role in the process of lymphocyte extravasation. The demonstration of lymphoid organ- and inflammation-specific microvascular antigens offers direct evidence for a complex specialization of endothelium in relation to immune stimuli, and supports the concept that microvascular differentiation may play an important role in local immune responses.     

Most B cells in non-lymphoid tissues are naïve

The current view of lymphocyte migration states that na?ve lymphocytes re-circulate between the blood and the lymph via the lymph nodes, but are not able to access non-lymphoid tissues. We examined B lymphocytes in peripheral tissues and found that the majority were phenotypically similar to na?ve B cells in lymphoid tissues and were located within the parenchyma, not associated with blood vessels. The mutation rate within the Vh region of these cells was substantially less than the rate attributed to somatic hypermutation and was identical to that observed in na?ve B cells isolated from the lymph nodes, showing the presence of na?ve B cells in the non-lymphoid organs. Further, using FTY720-treated mice, we showed that na?ve B cells migrate through the peripheral tissues and, using pertussis toxin, that the entry of B cells was not controlled by chemokine-mediated signalling events. Overall, these results show that na?ve B lymphocytes constitute the majority of the total B-cell population in non-lymphoid tissues and suggest that these cells may re-circulate through the periphery as part of their normal migration pathway. This has implications for the current view of the role of na?ve B cells in priming and tolerance.     

Cloak and dagger in the avoidance of immune surveillance

CD95 and CD95-ligand (CD95L) are physiological mediators of apoptosis required for the control of cell numbers in the human immune system. Discoveries in CD95-dependent mechanisms of immune evasion by tumours suggest regulation by oncogene expression. Clonal contraction of lymphocytes by a CD95/CD95L-independent mechanism has been reported and new evidence supports a role for CD95-dependent peripheral lymphocyte deletion by non-lymphoid tissue. Additionally, factors affecting the pro- and anti-inflammatory effects of CD95L point to a balance of cytokines and growth factors.     

Cellular and humoral immunity factors as regulators of regenerative morphogenesis

The published and author's data concerning changes in the immune system during regeneration of various organs are summarized. During the first few hours after partial removal of organs possessing high regeneration capacity, lymphocytes stimulate proliferation of nonlymphoid cells of an organ identical to the operated one; the production of antibodies against thymus-dependent antigen also increases. At the following stages of regeneration, the lymphocytes suppress the cell proliferation and decrease the antigen production. The level of these changes correlates with the level of post operational deficiency of the organ being maximal after total removal of the organ. The functional properties of splenocytes at different stages of regeneration suggest that the high T-helper and T-suppressor activities correlate with stimulation and suppression of non-lymphoid cells proliferation respectively. Culture medium supernatant after cultivation of these lymphocytes also changes the proliferation of hepatocytes. The author considers the impairment of natural immune tolerance caused by deficiency of organ auto-antigens that normally suppress lymphocyte proliferation to be the cause of the changes in lymphocyte activity.     

Ubiquitous expression of mRFP-1 in vivo by site-directed transgenesis

Progress in our understanding of the molecular cellular basis of immune function depends on our ability to track and image individual immune cells in vivo. To this end, the development of mouse models over-expressing various fluorescent proteins would represent an important experimental tool. In this report, we describe the generation and characterization of pUbi-mRFP-1 transgenic mice, in which the monomeric form of red fluorescent protein is ubiquitously expressed in various lymphoid and non-lymphoid tissues. Our newly generated pUbi-mRFP-1 mice are unique among previously reported mice transgenic for red fluorescent proteins because a single-copy of the mRFP-1 transgene driven by human ubiquitin C promoter has been integrated by homologous recombination into the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. We show that the distinct and uniform levels of mRFP-1 expression allow easy identification of transferred hematopoetic cells by FACS analysis or confocal microscopy, even when the transferred population represents a very small proportion in the target organ. Also, even in long-term experiments, we have seen no evidence of rejection of transferred pUbi-mRFP-1 lymphocytes. Due to its far-red spectrum, mRFP-1 is an ideal partner for dual imaging with green fluorescent proteins. We observed a good visual separation between donor lymphocytes derived from either mRFP-1 or eGFP transgenic mice in recipient animals. Our study suggests that the new pUbi-mRFP-1 transgenic mouse strain offers new opportunities for studying cellular interactions and migratory patterns of cells, especially for dual imaging of different cell types. In summary, our results demonstrate that a controlled strategy of transgenesis provides an effective means of ubiquitously expressing fluorescent proteins in vivo.     

Rate-limiting factors for lymphocyte protein synthesis. Ribosome commitment and the capacity of lymphocyte cell-free systems to translate exogenous mRNAs.

The properties of the protein synthesising systems of different lymphocyte preparations have been compared with those of non-lymphoid tissues. Polysome profiles from rat thymocytes, sheep mesenteric lymphocytes, rat liver and mouse ascites tumours showed that the commitment of ribosomes to protein synthesis in lymphocytes was relatively low. Initiation factor activities, assessed on the abilities of post-mitochondrial fractions to support exogenous mRNA translation, were limited or undetectable in lymphoid tissues. While the thymocyte system translated globin mRNA, the response was enhanced by ascites extracts rich in initiation factors. The mesenteric lymphocyte system responded only marginally to globin mRNA and poly(U) but the responses were not enhanced by ascites extracts. The activity of isolated mesenteric ribosomes was comparable with ribosomes from other tissues, indicating that extraribosomal factors were responsible for the poor overall activity of the mesenteric system. Finally, the effects of cycloheximide on the recruitment of polysomes in lymphocytes indicated that the commitment of ribosomes to protein synthesis might be restricted by both limited mRNA availability and limited capacity for initiation of mRNA translation.     

Expression profiling in mouse fetal thymus reveals clusters of coordinately expressed genes that mark individual stages of T-cell ontogeny

To search for genes that participate in regulatory networks sustaining mouse embryonic T-cell development, we have performed expression profiling using nylon macroarrays. Labeled samples representative of individual developmental stages were utilized, taking advantage of cell homogeneity during early thymus ontogeny. cDNAs revealing differential expression were further selected using labeled samples derived from lymphoid versus non-lymphoid tissues, and from mutant thymi exhibiting T-cell developmental defects. We thus identified clusters of coexpressed genes during T-cell embryogenesis and characterized their sequences through bioinformatics. We compare our results with those from other profiling analyses in the immune system, and discuss their implications for the definition of genes whose products are involved in T-cell development.     

2',5'-Oligoadenylate synthetase activity in lymphocytes from normal mouse.

The activity of 2',5'-oligoadenylate synthetase, an enzyme recently discovered in interferon-treated cells, was found in lymphocytes from normal mouse spleen that had received neither exogenous interferon nor its inducers. The oligoadenylate synthesized by lymphocyte cell extracts inhibited protein synthesis in rabbit reticulocyte lysates. The oligomers were composed mainly of trimer and were resistant to digestion by T2 ribonuclease. The level of the enzyme in lymphocytes was about 20 to 30% of that in L929 cells treated with interferon. The activity of the enzyme was further enhanced in lymphocytes in vitro by addition of interferon. The 2',5'-oligoadenylate synthetase was distributed among several lymphoid tissues, but was not detected in cell extracts from brain or liver. The enzyme may play an important role in the regulation of the immune system.     

Systemic Hematogenous Maintenance of Memory Inflation by MCMV Infection

Several low-grade persistent viral infections induce and sustain very large numbers of virus-specific effector T cells. This was first described as a response to cytomegalovirus (CMV), a herpesvirus that establishes a life-long persistent/latent infection, and sustains the largest known effector T cell populations in healthy people. These T cells remain functional and traffic systemically, which has led to the recent exploration of CMV as a persistent vaccine vector. However, the maintenance of this remarkable response is not understood. Current models propose that reservoirs of viral antigen and/or latently infected cells in lymph nodes stimulate T cell proliferation and effector differentiation, followed by migration of progeny to non-lymphoid tissues where they control CMV reactivation. We tested this model using murine CMV (MCMV), a natural mouse pathogen and homologue of human CMV (HCMV). While T cells within draining lymph nodes divided at a higher rate than cells elsewhere, antigen-dependent proliferation of MCMV-specific effector T cells was observed systemically. Strikingly, inhibition of T cell egress from lymph nodes failed to eliminate systemic T cell division, and did not prevent the maintenance of the inflationary populations. In fact, we found that the vast majority of inflationary cells, including most cells undergoing antigen-driven division, had not migrated into the parenchyma of non-lymphoid tissues but were instead exposed to the blood supply. Indeed, the immunodominance and effector phenotype of inflationary cells, both of which are primary hallmarks of memory inflation, were largely confined to blood-localized T cells. Together these results support a new model of MCMV-driven memory inflation in which most immune surveillance occurs in circulation, and in which most inflationary effector T cells are produced in response to viral antigen presented by cells that are accessible to the blood supply.     

Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells

The radiosensitive cell populations, such as resting lymphocytes from thymus, spleen, lymph node and blood, have much smaller nuclei (Vn (nuclear volume) approximately 20 to 70 microns3) compared to radioresistant G0 cells from non-lymphoid tissues (liver, kidney, brain, heart; Vn approximately 75 to 2700 microns3). It is suggested that radiation-induced disorganization of nuclear structures and cell pycnosis (interphase death) are promoted in G0 lymphocytes because in normal physiological conditions their nuclei assume a higher degree of chromatin condensation. In contrast, dispersion of chromatin into larger nuclear volumes, such as those of most non-lymphoid G0 cells, may hinder or delay radiation-induced cell death.     

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b^- DC, and CD11b⁺ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.     

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.     

Utility of antiPax5 in the diagnosis of lymphoproliferative disorders and neoplasia in mice

CD45R/B220 antigen (B220) is a common mouse panB-cell marker used for paraffin-embedded tissues. However, antiB220 has limited specificity in diagnostic pathology because the B220 antigen is expressed on subsets of cytotoxic T lymphocytes and natural killer cells, on plasmacytic dendritic cells, and on T lymphocytes of mice with the lymphoproliferative disorder associated with Fas (lymphoproliferative mutant mouse, B6.MRL-Fas(lpr/J)) or Fas ligand (generalized lymphoproliferative disease mutant mouse, C3H/ HeJ-Fasl(gld/J) or B6Smn.C3-Fasl(gld/J)). In addition, mouse B lymphocytes vary in the amount of B220 expressed, and some subsets of mouse B lymphocytes do not express B220 at all. In comparison, Pax5 expression (detected by immunohistochemistry using antiPax5) offers greater specificity and sensitivity because of its earlier expression during B-cell differentiation, its ability to detect all committed B cells, and its restriction to the B-cell lineage. Here we describe the use of an antibody to human Pax5 in diagnostic pathology with formalin-fixed, paraffin-embedded mouse tissue.     

Immunoneutralization of endometrial monoclonal nonspecific suppressor factor beta (MNSFbeta) inhibits mouse embryo implantation in vivo

Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.     

Effect of cytotoxic lymphocytes obtained by in vitro immunization with syngeneic lymphoma cells on pluripotent hemopoietic cells

The possibility of the presence of leukemia-associated antigens on pluripotent hemopoietic cells was studied with the aid of immune lymphocytes, cytotoxic against mouse syngeneic lymphoma cells. Cytotoxic lymphocytes were obtained during immunization in vitro of C57BL/6 mouse splenocytes by syngeneic T lymphoma EL-4 cells in the presence of interleukin-2. Specific cytotoxic activity of immune lymphocytes as regards EL-4 cells was not blocked by addition of normal bone marrow cells. Incubation of the bone marrow with immune killers did not lead to a decrease in the number of colony-forming units in the spleen. It was shown that using cytotoxic lymphocytes the total killing of lymphoma cells might be achieved in a mixture of bone marrow and lymphoma cells, whereas pluripotent precursor cells might be retained.     

Production of heterologous antibodies to T-killers of mice immunized against H-2 antigens

Rabbit antisera were obtained against cytotoxic small peritoneal lymphocytes (IPEL) of CBA (H-2k) mice immune to alloantigens C57BL/6 (H-2b) and to the enriched 5-day MLC cytotoxic blast lymphocytes (MLC--CL). After appropriate absorption by cells and tissues of intact mice the cytotoxicity of the sera was lost relative to normal lymphoid cells. The absorbed anti-CPL serum inhibited, in the presence of complement, the cytotoxic effect of CPL but not that of MLC--CL on 51Cr-labeled allogeneic macrophages. This inhibition was restricted by idiotypic and strain specificity. Conversely, the absorbed anti-MLC--CL serum inhibited the cytotoxic effect of both CPL and MLC--CL of various mouse strains, irrespective of their immunologic specificity. It is supposed that the effect of the anti-CPL serum is mainly caused by antibodies againts idiotypic determinants of the killer T receptors, whereas the effect of the anti-MLC--CL serum is due to antibodies against differentiation antigens of the proliferating lymphocytes.