Developmental potential of CD4-8- thymocytes. Peripheral progeny include mature CD4-8- T cells bearing alpha beta T cell receptor

We have used the intra-thymic transfer system to investigate the population dynamics of thymocyte and mature T cell subsets in the absence of continuing precursor input from the bone marrow. We have followed the development and life span of CD4+ and CD8+ thymocyte subsets and mature peripheral T cells from intra-thymically injected adult or fetal CD4-8- thymic precursors. Both precursor types proliferated, differentiated, and exported to peripheral lymphoid tissues alpha beta-TCR+CD4+8- and CD4-8+ progeny which formed a stable, long-lived component of the peripheral T cell pool. The production of phenotypically mature thymocytes and peripheral T cells occurred more rapidly from fetal CD4-8- precursors. CD4+8-:CD4-8+ ratios among peripheral progeny of intra-thymically-injected CD4-8- precursors were initially normal, but they steadily declined among progeny of the fetal precursors. Thus, there appear to be differences in the life span and/or proliferative capacity of mature T cells derived from embryonic vs adult progenitors. In addition to the predominant CD4+8- and CD4-8+ subsets of peripheral T cells, a minor (1 to 20%) population of Thy-1+CD3+4-8- T cells was identified among peripheral progeny of intra-thymically-injected CD4-8- thymocytes, as well as in lymph nodes of unmanipulated animals. A total of 20 to 34% of this subset expressed V beta 8+ TCR and the majority were CD5hi, Pgp-1+, and J11d-. The function and specificity of this newly identified population of thymically derived peripheral T cells remains to be investigated.     

Distribution of immune cells expressing CD3a, CD21 and S-100 protein markers in the porcine gut-associated lymphoid tissues.

The distribution of immune cells within the gut-associated lymphoid tissues (GALT) of swine is highly organized. The appearance of such cells could not be separated from the effects of age, weaning and exposure to environment. Here, we have examined the distribution patterns of a subset of CD3a+ T and CD21+ B cells as well as S-100 protein+ cells and secretory (s) IgA+ cells within GALT compartments (such as jejunal lamina propria = JLP, ileal Peyerís patches = IPP, and mesenteric lymph node = MLN) of juvenile 8-week-old conventionally reared pigs using either two monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) in the immunohistochemical staining techniques with avidin-biotin complex (ABC) or peroxidase-antiperoxidase complex (PAP), respectively. The most potent porcine T-cell marker--CD3 surface antigen--is expressed as CD3a epitope on ileal intraepithelial lymphocytes, and numerous lymphocytes in the extrafollicular areas of MLN and dome region of IPP. Conversely, the cells expressing CD21 surface molecules were only demonstrable in the interfollicular areas of MLN and in the germinal centers of IPP. A strong reaction to sIgA was displayed by the plasma cells in the lumen of crypts and those residing the lamina propria of jejunum and ileum. The S-100 protein+ cells were numerous in JLP around the crypts and in IPP of weaned pigs. Both applied mAbs proved to be useful reagents for phenotypic and functional analyses of porcine lymphoid cell subsets by the ABC technique. However, further investigation of the S-100 protein marker is needed to determine which (if any) subset of porcine CD3+ CD4- CD8+ T cells could be designated as orthologue of human CD8+ CD11b+ suppressor T cells.     

Maternal Short-Chain Fructooligosaccharide Supplementation Influences Intestinal Immune System Maturation in Piglets

Peripartum nutrition is crucial for developing the immune system of neonates. We hypothesized that maternal short-chain fructooligosaccharide (scFOS) supplementation could accelerate the development of intestinal immunity in offspring. Thirty-four sows received a standard or a scFOS supplemented diet (10 g scFOS/d) for the last 4 weeks of gestation and the 4 weeks of lactation. Colostrum and milk immunoglobulins (Ig) and TGFβ1 concentrations were evaluated on the day of delivery and at d 6 and d 21 postpartum. Piglet intestinal structure, the immunologic features of jejunal and ileal Peyer''s patches, and mesenteric lymph node cells were analysed at postnatal d 21. Short-chain fatty acid concentrations were measured over time in the intestinal contents of suckling and weaned piglets. Colostral IgA (P<0.05) significantly increased because of scFOS and TGFβ1 concentrations tended to improve (P<0.1). IFNγ secretion by stimulated Peyer''s patch and mesenteric lymph node cells, and secretory IgA production by unstimulated Peyer''s patch cells were increased (P<0.05) in postnatal d 21 scFOS piglets. These differences were associated with a higher proportion of activated CD25+CD4α+ T cells among the CD4+ helper T lymphocytes (P<0.05) as assessed by flow cytometry. IFNγ secretion was positively correlated with the population of activated T lymphocytes (P<0.05). Total short-chain fatty acids were unchanged between groups during lactation but were higher in caecal contents of d 90 scFOS piglets (P<0.05); specifically propionate, butyrate and valerate. In conclusion, we demonstrated that maternal scFOS supplementation modified the intestinal immune functions in piglets in association with increased colostral immunity. Such results underline the key role of maternal nutrition in supporting the postnatal development of mucosal immunity.     

Expression of stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4, on CD34+ human bone marrow cells is a phenotypic alteration for committed lymphoid progenitors.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.     

Identification and distribution of the costimulatory receptor CD28 in the mouse.

T cell activation requires Ag-specific stimulation mediated by the TCR as well as an additional stimulus provided by Ag presenting cells. On human T cells, it has been shown that antibodies to the Ag CD28 can provide a potent amplification signal for cytokine production and proliferation. Here we describe the production of a mAb to the murine homologue of CD28, and the use of this antibody to examine the function and distribution of CD28 in the mouse. Anti-murine CD28 synergizes with TCR-mediated signals to greatly enhance lymphokine production and proliferation of T cells, and the CD28 signal is not blocked by cyclosporin A. In the peripheral lymphoid organs and in the blood of the mouse, all CD4+ and CD8+ T cells express CD28. In the thymus, CD28 expression is highest on immature CD3-, CD8+ and CD4+8+ cells, and on CD4-8- cells that express alpha beta and tau delta TCR. The level of CD28 on mature CD4+ and CD8+ alpha beta TCR+ thymocytes is two- to fourfold lower than on the immature cells. The potent costimulatory function of CD28 on mature T cells, together with the high level of expression on CD4+8+ thymocytes, suggest that this costimulatory receptor might play an important role in T cell development and activation.     

Recirculatory and sessile CD4+ T lymphocytes differ on CD45RC expression

CD4+ T cell subsets are unequally distributed in rat secondary lymphoid organs. Those with the memory phenotype CD45RClow Thy-1- L-selectin- are present at a higher frequency in Peyer's patches (PP) than in lymph nodes and spleen, and increase in numbers with age in all three tissues, particularly in the PP. Homing experiments revealed that CD4+ T cells that recirculate through secondary lymphoid organs are mainly CD45RChigh. It was also apparent that the ability of recirculating cells to enter different lymphoid organs varies; less cells enter PP than the spleen or lymph nodes. Our results also reveal the existence of a nonrecirculating population of CD4+ T cells in secondary lymphoid organs, which are predominantly, if not exclusively, CD45RClow. Our results show that secondary lymphoid organs differ in their CD4+ T cell subset composition as a consequence of having different ratios of recirculatory:nonrecirculatory CD4+ T cells, and these cells display a different CD45RC phenotype.     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRbeta gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cbeta, and Id2; and show a surface marker pattern (CD44+ CD25- CD24+ CD5-) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Valpha and Vbeta genes as well as CD3epsilon, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the alphabeta T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-gamma after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.     

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RBhigh lymph node T cells: functional role for thyroid-stimulating hormone during immune activation

Thyroid-stimulating hormone (TSH), a central neuroendocrine mediator of the hypothalamus-pituitary-thyroid axis, has been shown to affect various aspects of immunological development and function. To gain a better understanding of TSH involvement within the mammalian immune system, the expression and distribution of the TSH receptor (TSHr) has been studied by immunoprecipitation and by flow cytometric analyses. Using highly enriched populations of B cells, T cells, and dendritic cells, trace amounts of TSHr were precipitated from B cells and T cells, whereas high levels of TSHr were precipitated from the dendritic cell fraction. Flow cytometric analyses of TSHr expression on splenic and lymph node T cells revealed a major difference between those tissues in that only 2-3% of splenic T cells were TSHr+, whereas 10-20% of CD4+8- and CD4-8+ lymph node T cells expressed the TSHr, which was exclusively associated with CD45RB(high) cells and was not expressed during or after activation. The TSHr was not present on cells of the immune system during fetal or neonatal life. However, recombinant TSHbeta was found to significantly enhance the phagocytic activity of dendritic cells from adult animals and to selectively augment IL-1beta and IL-12 cytokine responses of dendritic cells following phagocytic activation. These findings identify a novel immune-endocrine bridge associated with professional APCs and naive T cells.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

A murine thymic stromal cell line which may support the differentiation of CD4-8- thymocytes into CD4+8- alpha beta T cell receptor positive T cells.

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4-8- (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4+CD8- cells were maintained further while the CD4+8+ cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor alpha beta. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of alpha beta lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.     

Age Associated Changes in the Distribution of Ipr Gene-Induced B220-Positive T Cells in Lymphoid Organs of MRL/Mp-Ipr/Ipr Mice Using Dual Exposure Microphotographs of Double Immunofluorescence Staining

Homozygous MRL/Mp-1pr/1pr (MRL/1pr) mice, which have an autosomal recessive mutant 1pr gene and exhibit defects in Fas antigen, spontaneously develop autoimmune disease with progressive expansion and accumulation of characteristic abnormal CD4-CD8-double negative T cells that express B220 surface antigen, a B cell-specific surface marker in normal mice. We analyzed the distribution and age related changes of lpr gene-induced abnormal T cells (B220-positive lpr T cells) In the lymphoid organs of MRL/1pr mice. We studied cryostat sections of the spleen, peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches at different stages using FITC (fluorescein isothiocyanate)-conjugated monoclonal antibodies directed against B220 (RA3-6B2) and PE (phycoerythrin)-conjugated anti-mouse CD3 (2C11) monoclonal antibody, examining dual-exposure microphotographs of double-immunofluorescence stained preparations. We observed that in aged MRL/lpr mice, B220-positive abnormal 1pr T cells were not present in the thymus-dependent area, and the majority of the follicular area cells were displaced by 1pr T cells. These findings suggest that the cellular trafficking of B220-positive lpr T cells differs from that of conventional T cells and that these 1pr-derived T cells play a role in the follicle.     

Development of T lymphocytes at extrathymic sites.

T lymphocytes expressing both CD4 and CD8 are the predominant cell type in the thymic cortex but are extremely rare outside the thymus of normal mice. In this article, we show that if precursor thymocytes (CD4-CD8-) from fetal or adult donors are injected i.v. into irradiated recipients, some of these cells will lodge in lymph nodes and develop into both CD4+CD8+ (double-positive) and CD4+ or CD8+ (single-positive) cells. This phenomenon also occurred in thymectomized recipients, strongly suggesting it is genuine extrathymic development. Prethymic precursors (e.g., fetal liver), were unable to use the lymph node for T cell development, without thymic processing. The data suggest that given unusual circumstances (irradiation or thymectomy and availability of appropriate precursors), the lymph nodes can support T cell development.