Specific in vivo suppression of lymph node cell proliferation and humoral immune responses by cloned antigen-specific T suppressor cells

Two BSA-specific Ts cell clones have been isolated from CBA/J mice tolerized by low doses of BSA. Together with one Ts cell clone isolated from an immune animal, they have recently been characterized with regard to phenotypes and in vitro functions. In the present report the in vivo effector functions of two of them (Ts cell clones BVI/5 and HF1.MS) are described. BSA-primed lymph node cells from CBA/J mice, which had received BVI/5 or HF1.MS Ts cells at the time of immunization, do not respond to a subsequent in vitro antigenic challenge. A human gamma-globulin (HGG)-specific lymph node cell proliferation is not influenced. BVI/5 Ts cells injected into mice at the time of priming with fluorescein (Flu)-conjugated BSA or Flu-HGG inhibit the humoral anti-Flu-response in Flu-BSA-primed animals. The anti-Flu-response in Flu-HGG-primed animals is only marginally affected by BVI/5 Ts cells. The data show that it is possible to induce immunologic unresponsiveness at the humoral level by reexposing in vitro propagated Ts cell clones to their syngenic in vivo environment.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Adult murine lymph node cells respond blastogenically to a new differentiation antigen on isologous and autologous B lymphocytes.

Cells derived from lymph nodes (LN) of adult CBA mice respond blastogenically to mitomycin-treated autologous, as well as isologous spleen cells. This isogeneic LN-to-spleen (mixed lymphocyte culture) is best obtained when both responder and stimulator cells are derived from donors greater than 10 weeks of age. Responsive cells appear restricted to LN since they could not be detected in adult spleen, marrow, or thymus. LN cells do not require the presence of spleen in order to differentiate into responder cells since those derived from neonatally splenectomized mice are fully active. Stimulator cells appear in the spleen, bear Ig on their surfaces, and can be detected in spleens of irradiated, bone marrow-reconstituted mice. Experiments comparing the responsiveness of adult LN cells and that of neonatal T cells toward mitomycin C-treated lymphoid cells from a variety of sources suggest the presence of two iso-antigens on B lymphocytes. Since both antigens apparently are absent on precursor bone marrow cells and develop with time, they have been classified as murine differentiation antigens 1 and 2 (MDA-1, MDA-2). Whereas both appear in the spleen, only one, MDA-1, is also detectable by this methodology in LN. Both MDA-1 and MDA-2 activate neonatal T cells, but MDA-2 triggers only adult LN. Whereas MDA-2 developed in an x-irradiated, bone marrow-reconstituted spleen, MDA-1 did not over a 9-week interval.     

Popliteal lymph node enlargement induced in syngeneic hosts by T cells from foster-nursed mice

Splenocytes from adult F1 mice were assayed for their capacity to induce popliteal lymph node enlargement (PLNE) when inoculated in the footpad of identical or reciprocal F1 hosts. The results obtained showed that: (i) T Lyt 1+ splenocytes from adult F1 hybrids were able to induce a significant PLNE when inoculated in reciprocal but not in identical F1 hosts. (ii) Foster-nursing of F1 hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their T splenocytes to induce PLNE: Lyt 1+ splenocytes were able to induce significant PLNE in identical but not in reciprocal F1 hosts. Thus, the ability of T splenocytes from foster-nursed F1 hybrids to induce PLNE resembled that observed in reciprocal F1 hybrids nursed by their own mothers. (iii) PLNE was accompanied by cell proliferation involving host B and T lymphocytes. (iv) This PLNE could be detected using F1 hybrids from parental strains differing or not at H-2 antigens but involving a parental strain expressing the stimulatory Mlsa allele and a parental strain bearing the nonstimulatory Mlsb allele while it was not observed in F1 hybrids from parental strains sharing Mlsa antigens.     

Incomplete differentiation of antigen-specific CD8 T cells in tumor-draining lymph nodes

CD8 T cells lacking effector activity have been recovered from lymphoid organs of mice and patients with progressing tumors. We explored the basis for lack of effector activity in tumor-bearing mice by evaluating Ag presentation and CD8 T cell function in lymphoid organs over the course of tumor outgrowth. Early after tumor injection, cross-presentation by bone marrow-derived APC was necessary for T cell activation, inducing proliferation and differentiation into IFN-gamma-producing, cytolytic effectors. At later stages of outgrowth, tumor metastasized to draining lymph nodes. Both cross- and direct presentation occurred, but T cell differentiation induced by either modality was incomplete (proliferation without cytokine production). T cells within tumor-infiltrated nodes differentiated appropriately if Ag was presented by activated, exogenous dendritic cells. Thus, activated T cells lacking effector function develop through incomplete differentiation in the lymph nodes of late-stage tumor-bearing mice, rather than through suppression of previously differentiated cells.     

Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and na?ve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production of IL-6 and tumour necrosis factor-alpha to bacteria, which elicited mainly IL-10 in monocyte-derived DC. In contrast, comparable IFN-gamma production patterns were found in both natural killer cells and T cells induced by all bacteria-matured APC. An inhibitory effect of certain strains on this IFN-gamma production was also mediated by all types of APC. The most potent responses were induced by monocyte-derived DC, which thus constitute a sensitive screening model.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Cellular events in the lymph node and lung of mice with influenza. Consequences of depleting CD4+ T cells.

The cellularity of the mediastinal lymph nodes of mice infected intranasally with a high dose of an H3N2 influenza A virus increases massively within 5 days. All classes of lymphocytes are involved. A similar, but much smaller, expansion in cell numbers occurs after exposure to a comparable dilution of normal chick allantoic fluid. In the control group, this increase in lymph node size is totally prevented by the in vivo depletion of CD4+ T cells whereas there is only a 50% reduction in the virus-infected mice. The lymphocyte component of the cellular exudate in the lungs of infected mice is dominated by activated, CD8+ T cells, which are also prevalent in the mediastinal lymph nodes. Elimination of the CD4+ subset does not greatly diminish the severity of this inflammatory process. The CD4-depleted mice clear the virus from the lung, and there is little effect on the frequency of virus-specific, cytotoxic T lymphocyte precursors in either the lymph node or the lung. Substantial involvement of CD4+ T cells is not essential for the development of effective cell-mediated immunity in mice with influenza.     

CD4 T cells activated in the mesenteric lymph node mediate gastrointestinal food allergy in mice

A localized Th2 milieu has been observed in the intestine of subjects with food allergic disorders; however, the role of T cells in the pathophysiology of these disorders remains poorly understood. Our aim was to examine sites of T cell activation in response to food challenge, identify potential factors responsible for T cell recruitment to the gut, and determine the role of T cells in disease. BALB/c mice were systemically sensitized to ovalbumin (OVA) and repeatedly fed with OVA to induce allergic diarrhea. Local cytokine and chemokine expressions were assessed by quantitative PCR, and cytokine secretion levels in the mesenteric lymph node (MLN) were determined by ELISA. Homing molecule expression was determined by flow cytometry, and the role of CD4(+) T cells in promoting disease was tested by adoptive transfer. Mice developed diarrhea associated with changes in epithelial ion transport, mast cell infiltration, intestinal IgE secretion, and local upregulation of Th2 cytokines and the Th2 chemokines CCL1, CCL17, and CCL22 in the small intestine. T cell activation occurred in the MLN before symptom onset, and a single feed of OVA induced T cell proliferation, alpha(4)beta(7) upregulation, and CD62L downregulation. Cells from the MLN, including purified CD4(+) T cells, were able to transfer allergic diarrhea to naive mice. A gut-homing phenotype induced in the MLN and selective upregulation of Th2 chemoattractants are likely important factors in the gastrointestinal recruitment of pathological Th2-skewed CD4(+) T cells in food allergy.     

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF-MF). We report that ELF-MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re-established the apoptotic response to ELF-MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF-MF.     

Frequency of herpes simplex virus-specific helper T lymphocyte precursors in the lymph node cells of infected mice

We have established a limited dilution assay to estimate the frequency of herpes simplex virus type 1 (HSV)-specific, interleukin 2 (IL 2)-producing helper T lymphocyte precursors (HTL-P). The estimated frequency of such cells in suspensions of local lymph node (LN) cells 5 days after in vivo virus infection was 1:2470 to 1:5800. Frequencies of HTL-P in cells from uninfected mice were below levels of detection of our system and were judged to be below 1:100,000. Removal of Lyt-2+ cells from responder LN cells before culture increases HTL-P frequency twofold to threefold, indicating the likely operation of some form of suppression in unseparated cultures. The demonstration of HSV-specific HTL-P required that cells from virus-primed mice be reexposed in vitro to viral antigen. In addition, clones expanded during a 9-day culture period failed to generate IL 2 unless reexposed to specific viral antigen or cross-reactivate HSV-2. Thus, HSV-specific HTL-P were strictly antigen dependent. No evidence was obtained for antigen-independent subpopulations of HTL-P as occurs with viral-specific cytotoxic T lymphocyte precursors. The clonal progeny of HTL-P were of the Thy-1+ Lyt+2- phenotype. Priming in vivo for the subsequent in vitro detection of HTL-P required that mice be exposed to infectious virus. Thus neither UV-inactivated nor heat-inactivated nor extracted viral glycoproteins could prime for HTL-P detection. The relevance of these findings for the future use of subunit vaccines against HSV is briefly discussed.     

Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice.

An in vitro assay which measures antigen-induced proliferation of primed murine lymph node cells is described. The response is mediated by T eclls since it can be obtained by using nylon wool-passed lymphocytes (less than 1% Ig+ cells) and it can be abolished by treatment with anti-Thy 1.2 and C. Furthermore, LN cells from nu/nu mice injected with antigen do not demonstrate antigen-induced proliferation in contrast to the response observed in euthymic littermate controls. Other relevant parameters of this proliferative assay include the observations that the response is highly antigen specific, can be seen as early as 4 days and as late as 60 days after in vivo priming, is restricted to the use of certain sets of LN when animals are injected subcutaneously at the base of the tail, and can be seen with LN cells from mice primed with antigen in either CFA or ICFA. The ease of the assay coupled with its specificity and quantitative dimensions provides a direct and simple method to evaluate processes involved in antigen-induced murine T lymphocyte activation.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Homing of antigen-specific T cells in the Lewis rat. I. Accumulation of antigen-reactive cells in the perithymic lymph nodes

A permanent ovalbumin-specific T cell line of "helper/inducer" cell phenotype (W 3/13+, W 3/25+, OX 8-) was used to study the homing pattern in normal untreated Lewis rats. After i.v. injection, the migration of these cells was followed directly by using 51Cr- or [14C]thymidine-labeled cells. In addition, I tried to retrieve the cells from different lymphatic tissues by antigen restimulation. I found that most of the radiolabeled cells migrate to the lung, liver, kidney, and spleen. Other lymphoid tissues such as the thymus and the cervical and mesenteric lymph nodes were almost devoid of such cells with one exception: the perithymic lymph nodes (pt-LN). Twenty-four hours after the cell transfer, viable antigen-specific cells could be recovered from these organs. Within 9 days the pt-LN enlarged, the percentage of W 3/13+ and W 3/25+ T lymphocytes was enhanced, and both relatively high spontaneous and antigen-driven responses were measurable in cell cultures of these lymph nodes. All the effects were observed if viable but not irradiated antigen-specific T blasts were transferred. Moreover, after active immunization, antigen-reactive cells appeared to accumulate not only in the draining but also in the pt-LN. In both experimental situations, the adoptive transfer and the in vivo activation of antigen-specific lymphocytes, the pt-LN appear to play an important role in the homing of such cells.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

Antigen-specific lysis in vitro of lymph node cells of intact mice injected with RNA from viable lymph node cells of immunized animals]

Supernatant fluid obtained after centrifugation of the suspension of viable lymph node cells of immunized animals proved to induce in vivo in the lymph node cells of intact mice sensitivity to lysis with a specific antigen in vitro. This property was possessed after chromatography of the supernatant fluid on Sephadex G-200 by the 3rd fraction (MW about 30000 dalton). DNA-ase, trypsin or deproteinization failed to influence whereas RNA-ase inactivated this fraction in respect to the inducing properties.     

Culture-independent identification of microorganisms that respond to specified stimuli.

A new approach that permits culture-independent identification of microorganisms that respond to specified stimuli was developed. This approach was illustrated by examination of microorganisms that grew in response to various nutrient supplements added to soil. A thymidine nucleotide analog, bromodeoxyuridine (BrdU), and supplements were added to soil and incubated for 3 days. DNA was extracted from the soil, and the newly synthesized DNA was isolated by immunocapture of the BrdU-labeled DNA. The unique perspective this approach offers was demonstrated by comparing the microbial community structures obtained from total soil DNA and the BrdU-labeled fraction in an rRNA gene (rDNA) analysis. The traditional total DNA analysis revealed no notable differences between the treatments, whereas the BrdU-labeled DNA showed significantly different banding patterns between the nutrient supplement treatments and compared with total DNA banding patterns. PCR primers were developed to specifically amplify the intergenic region of an rDNA sequence unique to the BrdU analysis of a phosphate supplement treatment. Amplification of DNA from all treatments using these primers showed that it was unique to the phosphate treatment and that it was present in both the total DNA and BrdU-labeled DNA fractions. This result demonstrates the promise of this new strategy, because it was able to permit identification of a sequence from a phosphate-responsive organism that was not discernable in the traditional total DNA community structure analysis.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.