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.     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

T lymphocytes that express CD4 and the alpha beta-T cell receptor but lack Thy-1. Preferential localization in Peyer's patches

Thy-1- T cells expressing CD4 and the alpha beta-TCR have been identified in murine lymphoid tissues. These cells are particularly prevalent in Peyer's patches (PP), representing 17 +/- 3% of PP CD4 T cells, whereas they are much less prevalent in spleen, lymph nodes, lamina propria, or peritoneum. Phenotypic studies of fresh-isolated PP T cells demonstrate that all PP CD4 T cells (both Thy-1- and Thy-1+) express CD3, alpha beta-TCR, and CD5 (Lyt-1), whereas none coexpress CD8 (Lyt-2). Thy-1- and Thy-1+ CD4 T cell lines generated from PP also coexpress CD3 and alpha beta-TCR, but are heterogeneous in expression of CD5 and again do not coexpress CD8. Further studies revealed that Thy-1- CD4+ T cells were not present in nude mice. Short term stimulation of Thy-1+ CD4+ PP T cells with anti-CD3 resulted in loss of Thy-1 in a substantial fraction of these cells. Functional studies of Thy-1- and Thy-1+ CD4+ PP T cells indicate that fresh-isolated Thy-1- CD4+ cells do not proliferate in response to insoluble anti-CD3 but do proliferate when stimulated with soluble anti-CD3 in the presence of feeder cells. In contrast, Thy-1+ CD4+ cells proliferate well to both stimuli. However, Thy-1- CD4+ PP T cells adapted to in vitro culture exhibit vigorous proliferative responses when stimulated with either form of anti-CD3. Evaluation of lymphokine secretion by fresh-isolated Thy-1- and Thy-1+ CD4+ PP T cells revealed that both make substantial amounts of IL-2; however, Thy-1- T cells made less IL-4 than their Thy-1+ counterparts. Neither population made IL-5 or IFN-gamma. Similarly, Thy-1- and Thy-1+ CD4 T cell lines made similar amounts of IL-2; again Thy-1- T cells made less IL-4; and in this case Thy-1- T cells made IL-5 albeit significantly less than the Thy-1+ cells. Finally, immunohistochemical studies suggested that many of the CD4+ T cells in PP germinal centers were Thy-1-, indicating that Thy-1- and Thy-1+ CD4 T cells differ in their distribution within the PP. These studies thus define a phenotypically and functionally distinct T cell population which is most prevalent in murine Peyer's patches.     

Localization of PNA-binding and nonbinding thymus cells in peripheral lymphoid organs

Thymus cells were labeled in vitro with FITC and injected into syngeneic recipients. In cell suspensions of lymphoid organs green cells were inspected for PNA receptors with double immunofluorescence. A striking preference of PNA-negative cells to localize in lymph nodes and the lymphoid compartment of the spleen was demonstrated. Incubation with anti-Ly sera revealed that Ly 1⁺ PNA-negative cells homed in popliteal lymph nodes and Peyer's patch but not in mesenteric lymph nodes.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.     

Antigen presentation by macrophage-enriched cells from the mouse Peyer's patch

Macrophage-enriched cells derived from Peyer's patches were prepared with or without preincubation of whole patches in collagenase-containing medium. The cells thus obtained were pulsed with ovalbumin (OVA), added to OVA-primed whole lymph node (LN) cells or LN T cells and proliferative responses stimulated in the latter cells were assessed after 5 days by thymidine incorporation. Macrophage-enriched cells obtained from Peyer's patches (PP) preincubated with collagenase presented antigen as well or better than macrophage-enriched cells obtained from spleens. In contrast, macrophage-enriched cells obtained from PP which were not preincubated with collagenase were frequently unable to function as antigen-presenting cells. In further studies, macrophage-enriched cells obtained from mice which had been fed OVA were found to be capable of stimulating antigen-primed LN T cells in an antigen-specific manner without further exposure to antigen in vitro. These results indicate PP macrophage-enriched cells are fully capable of supporting the induction of immune responses in vitro and that macrophages from PP of orally primed mice may present antigen without further antigen exposure. Thus macrophages in PP may participate in the induction of immune responses to gastrointestinal antigens in vivo.     

The Qa-1 alloantigens. I. Identification and molecular weight characterization of glycoproteins controlled by the Qa-1a and Qa-1b alleles

Splenocytes from the Qa-Tla congenic strain pairs, A and A-Tlab or B6 and B6-Tlaa, were biosynthetically labeled with 3H-amino acids or cell surface labeled with 125I. Membrane proteins were solubilized with detergent and chromatographed on lentil lectin-Sepharose, and the resulting adherent pools were immunoprecipitated with antisera specific for determinants controlled by the Qa-1a and Qa-1b alleles, Qa-1.1 and Qa-1.2, respectively. Polyacrylamide gel electrophoresis analysis of immunoprecipitates from biosynthetically labeled preparations indicated that both the Qa-1.1 and Qa-1.2 antigens were glycoproteins with a m.w. of approximately 46,000. Qa-1.2 isolated from radioiodinated spleen cells similarly had a m.w. of 46,000. Analysis of anti-Qa-1.1 precipitates from 125I-labeled Qa-1a lysates demonstrated in addition to the 46,000 m.w. component, an electrophoretically heterogeneous protein or series of proteins in the m.w. range of 55,000 to 75,000. The specificity of these reactivities was shown by both antiserum and genetic control immunoprecipitations. These findings indicate that the Qa-1.1 and Qa-1.2 antigens are cell surface glycoproteins that are distinct from the TL antigens, and suggest a further complexity at the Qa-1--Tla locus.     

Systemic Antibodies Can Inhibit Mouse Mammary Tumor Virus-Driven Superantigen Response in Mucosa-Associated Lymphoid Tissues

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer’s patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer’s patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer’s patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer’s patches or NALT.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

Distribution of Mouse Adenovirus Type 1 in Intraperitoneally and Intranasally Infected Adult Outbred Mice

In situ nucleic acid hybridization and immunohistochemistry were used to determine the histological localization of mouse adenovirus type 1 (MAV-1) during acute infection of adult mice infected either intraperitoneally or intranasally with 1,000 PFU of wild-type virus. Organ samples were collected from days 1 to 17 postinfection for the intraperitoneally infected mice and from days 1 to 13 for the intranasally infected mice. Endothelial cells of the brain and spinal cord showed extensive evidence of MAV-1 infection. Endothelial cells in lungs, kidneys, and other organs were also positive for MAV-1, indicating a widespread involvement of the systemic circulation. The presence of viral nucleic acid and/or antigen was also demonstrated in lymphoid tissue. The spleens, Peyer’s patches, and peripheral lymph nodes showed positive staining at various times postinfection in mice infected by either route. Virus-infected cells in the spleen exhibited a stellate shape and were localized to the red pulp and germinal centers, suggesting that they are cells of the mononuclear phagocytic system.     

Characterization of determinants encoded by four Qa-1 genotypes and their recognition by cloned cytotoxic T lymphocytes

The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1a- and for Qa-1b-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2r and H-2f strains provided the strongest evidence to date for the designation of the Qa-1c and Qa-1d genotypes, respectively, for these strains. Qa-1c-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1b target cells, thus demonstrating that these antigens lack a Qa-1b-associated determinant. Similarly, Qa-1d encoded antigens were recognized by only half of the CTL clones that lysed Qa-1a target cells. In addition, one CTL clone that was cytotoxic for Qa-1b and Qa-1c target cells demonstrated low affinity, cross-reactive recognition of a Qa-1d encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1a, Qa-1b, and Qa-1d each encode multiple determinants. Two Qa-1d encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1a strains expressed indistinguishable Qa-1.1 antigens and the Qa-1b strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Variations in the expression of theta-C3H alloantigen on thymic and peripheral lymphocytes of newborn and young adult mice

The relative frequency of lymphocytes of mice showing varying degrees of surface θ-positivity (circumferential fluorescence) was recorded. Thymocytes were nearly 100% θ-positive. The relative proportions of θ-positive cells in Peyer's patches and lymph nodes of newborn mice varied in an almost identical fashion as a function of age. At 4 weeks of age and beyond, the relative numbers of θ-positive cells in Peyer's patches were consistently lower than in lymph nodes. As opposed to the predominance of thymocytes with complete rings, peripheralized thymic (T) lymphocytes showed a broad, age- and organ-dependent range of surface θ-positivity. These results suggest that surface θ may be lost rather rapidly upon emigration of lymphocytes from the thymus and/or that many θ-positive T cells with complete rings disappear within a short time. Variations in the relative proportion of complete rings on mesenteric lymph node cells on Days 1 and 4, were tentatively related to antigen-induced changes in the magnitude of thymocyte emigration.The pattern of surface θ-antigen of a given thymocyte or T cell with its size and DNA synthetic activity was compared. The findings suggest that incomplete ring fluorescence may especially be observed on proliferating lymphoblasts in the outer thymic cortex on their way to acquire the full complement of θ-antigen, and on medullary thymic lymphocytes or T cells having reentered mitotic activity, in response to antigenic and/or other microenvironmental stimuli. Our study yielded data consistent with the hypothesis that the progressive loss of surface θ-antigen does not represent a fully autonomous time-dependent process. Moreover, it is not clear if continued loss of θ-antigen by T cells below a certain threshold would render these cells undetectable by anti-θ sera.     

Removal of lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: effects on mitogen responses and evidence that ThB and certain Qa antigens are membrane-anchored via phosphatidylinositol

We reported previously that the Thy-1 antigen was released from murine thymocytes and thymoma cells by S. aureus-derived phosphatidylinositol-specific phospholipase C (PI-PLC). It is therefore part of a small group of proteins known to use a unique form of membrane attachment. This finding has now been extended in studies with peripheral lymphocytes and additional leukocyte markers. Retention of viability and responsiveness to LPS were excellent in PI-PLC-treated spleen cells and there was no appreciable effect on lectin-binding surface glycoproteins. Thy-1 regeneration was insignificant on unstimulated spleen cells within 24 hr of treatment, but nearly complete at this time with a continuously dividing cell line. In contrast to the result with LPS, responses to the mitogens Con A, PHA, and PWM were virtually eliminated. Of more than 40 monoclonal antibodies tested, only staining with ThB and particular Qa specificities were diminished by PI-PLC treatment. The latter included Qa-2, Qa-4, Qa-5, and possibly also Qa-6, whereas Qa-1, TLa, and other class I and class II histocompatibility antigens were unaffected. Although the validity of the Qa results seems assured by the total PI-PLC resistance of many other lymphocyte antigens, the pattern of release was notably different from that observed with Thy-1 and ThB. That is, the density of Qa-2 was usually unchanged on a subpopulation of Qa-2-positive cells. This raises interesting questions about lymphocyte heterogeneity and flexibility in the use of this form of surface protein anchoring. Glycosyl-phosphatidylinositol-linked proteins may be functionally significant in immunological responses, and this experimental approach should continue to be valuable for their identification and characterization.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Ontogeny of murine T lymphocytes: I. Maturation of thymocytes induced in vitro by tumor necrosis factor-positive serum (TNF+)

One question which is unresolved in developmental immunology is whether cortical thymocytes are the precursor cells which give rise to medullary thymocytes and peripheral T cells. Cortical thymocytes display a characteristic surface antigen phenotype (high TL and Thy-1, low H-2, no Qa-2, no Qa-3), are agglutinated by peanut agglutinin (PNA), and are unresponsive to concanavalin A (Con-A). The functionally more mature medullary thymocytes express a surface phenotype more closely resembling peripheral T cells (no TL, low Thy-1, high H-2, and some Qa-2), are not agglutinated by PNA, and are responsive to Con-A. An in vitro induction system has been devised in which mouse thymocytes undergo quantitative changes in surface antigens in less than 24 hr and increase their mitogen response to Con-A. The phenotypic changes are characterized by a decrease of TL and Thy-1 and an increase in H-2, Qa-2, and Qa-3. Studies in which thymocytes were fractionated on BSA gradients and by PNA agglutination demonstrate that the inducible cells have the properties of cortical thymocytes. Our data show that a subpopulation of cortical thymocytes can acquire phenotypic characteristics similar to medullary thymocytes and peripheral T cells.     

Radiation-induced alterations in murine lymphocyte homing patterns: I. Radiolabeling studies

In vitro X-irradiation of ⁵¹Cr-labeled spleen, lymph node, bone marrow, or thymus cells was found to alter their subsequent in vivo distribution significantly in syngeneic BDF₁ mice. Irradiated cells demonstrated an increased distribution to the liver and a significantly lower retention in the lungs. Cells going to the lymph nodes or Peyer's patches showed a significant exposure-dependent decrease in homing following irradiation. Irradiated lymph node cells homed in greater numbers to the spleen and bone marrow, while irradiated cells from other sources showed no preferential distribution to the same tissues. Sampling host tissues at various times after irradiation and injection did not demonstrate any return to normal patterns of distribution. The alterations in lymphocyte homing observed after in vitro irradiation appear to be due to the elimination of a selective population of lymphocytes or membrane alterations of viable cells, and the detection of these homing changes is in turn dependent upon the relative numbers of various lymphoid subpopulations which are obtained from different cell sources. Radiation-induced alterations in the normal homing patterns of lymphoid cells may thus be of considerable importance in the evaluation of subsequent functional assays in recipient animals.     

Characterization of cytotoxic cells generated from bone marrow culture

Bone marrow (BM) harbors precursors (Pre-NK) to NK cells. Recently, we devised an in vitro culture system that induces differentiation of the presumptive BM Pre-NK cells into cytotoxic cells to YAC in the presence of rat concanavalin A (Con A) conditioned medium. We have now analyzed the antigenic phenotype of the effector cells, precursor cells, and the target specificity of these cytotoxic cells. The cytotoxic cells had antigenic profiles similar to endogenous NK cells with the exception of Lyt-2 antigen. They are strongly positive for Qa-5, Thy-1, and partially positive for NK-1, Ly-5, Ly-6, Ly-10, and AsGm-1 and Lyt-2 antigens. The Pre-NK or accessory cells are positive for Qa-5, Ly-10, and Ly-20 and partially positive for NK-1, Thy-1, and AsGm-1 antigens. These Qa-5+ NK cells do not exhibit cytotoxic activity to WEHI or P815. They could also be generated from BM of nude mice as well as beige mice. We concluded from these studies that rat Con A-conditioned medium contained factors that could differentiate Pre-NK cells to mature NK cells and that these cells are heterogeneous. This in vitro culture system is useful in delineating the ontogeny of NK cells.