Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.     

Reciprocal T cell responses in the liver and thymus of mice injected with syngeneic tumor cells.

We investigated the T cell responses in various tissues, especially in the liver and thymus, of mice injected with syngeneic tumors. This study was undertaken since recent evidence indicated that the liver is one of the important immune organs for T cell proliferation. When C3H/He mice were intraperitoneally injected with mitomycin-treated syngeneic MH134 tumors (1 x 10(7)/mouse), a transient increase of liver mononuclear cells (MNC) was induced, showing a peak at Day 4 after injection. Histological study of such liver showed a sinusoidal dilatation and an accumulation of MNC in the sinusoids. The most predominant MNC induced were double negative (CD4-8-) alpha beta T cells and gamma delta T cells. These gamma delta T cells varied, showing unique time-kinetics. Despite a continuous increase of whole liver MNC and alpha beta T cells, the proportion of gamma delta T cells in the liver decreased beginning 4 days after injection. In contrast with the response in the liver, a striking decrease in the cell number of thymocytes was induced after tumor injection, showing a basal level at Day 6. This hypocellularity in the thymus appears to be an inverted response of the lymphocytosis in the liver. At this time, a corresponding decrease in the proportion of double positive (CD4+8+) T cells was always seen in the thymus. Analysis of cell proliferative response showed that the increase of liver MNC after tumor injection was accompanied by augmented proliferation, whereas the decrease of thymocytes was accompanied by depressed proliferation. The present results indicate that there exists a unique, reciprocal response of T lymphocytes between the liver and thymus, and that the presence of tumor appears to stimulate T cell response in the liver but alternatively inactivates such response in the thymus.     

Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen

The intestinal epithelium is comprised of a monolayer of intestinal epithelial cells (IEC), which provide, among other functions, a physical barrier between the high Ag content of the intestinal lumen and the sterile environment beyond the epithelium. IEC express a nonclassical MHC class I molecule known as the thymus leukemia (TL) Ag. TL is known to interact with CD8αα-expressing cells, which are abundant in the intestinal intraepithelial lymphocyte compartment. In this report, we provide evidence indicating that expression of TL by IEC modulates the cytokine profile of CD4(+) T cells favoring IL-17 production. We show in an adoptive transfer model of colitis that donor-derived cells become more pathogenic when TL is expressed on IEC in recipient animals. Moreover, TL(+)IEC promote development of IL-17-mediated responses capable of protecting mice from Citrobacter rodentium infection. We also show that modulation of IL-17-mediated responses by TL(+)IEC is controlled by the expression of CD8α on CD4(+) T cells. Overall, our results provide evidence for an important interaction between IEC and CD4(+) T cells via TL, which modulates mucosal immune responses.     

Activation and function of an autoreactive T cell clone with dual immunoregulatory activity

Previously it was demonstrated that the human autoreactive CD4+ T cell clone MTC-4 is bifunctional, having the capacity to augment differentiation of autologous B cells into Ig-secreting cells in the absence of PWM and the capacity to suppress such differentiation in the presence of PWM. In the present study it was shown that these two functions of MTC-4 are mediated by distinctly different mechanisms. In the presence of autologous class II MHC Ag, MTC-4 releases one or more non-MHC-restricted soluble factors which stimulate B cell differentiation. The helper factors are different from IL-2, and act on both resting (small) and activated (large) B cells. The suppressor function of MTC-4 cells is elicited when MTC-4 cells are co-cultured with autologous non-T cells preincubated with PWM for 4 h, but not with non-T cells preincubated with PWM for 24 h; thus, activated autologous non-T cells have a transient capacity to induce MTC-4 suppressor function. Induction of MTC-4 suppressor activity is not associated with increased proliferation of MTC-4 and is mediated by low numbers of these cells. Unlike helper function, MTC-4 suppression of Ig synthesis can occur late in B cell cultures, and MTC-4 suppresses Ig production by autologous B cells, but not by allogeneic B cells. Finally, in co-cultures with activated autologous non-T cells and allogeneic B cells, MTC-4 can simultaneously produce helper factors that augment Ig synthesis by allogeneic B cells and suppress Ig synthesis by autologous B cells. In summary, exposure of MTC-4 to autologous non-T cells causes release of non-MHC-restricted factors which augment Ig production by both resting and activated autologous B cells, whereas exposure of MTC-4 to recently activated B cells causes MTC-4 to express the additional function of directly suppressing Ig production by differentiated autologous B cells. Thus autoreactive T cells may be uniquely suited to regulate Ig production.     

Marginal zone B cells regulate antigen-specific T cell responses during infection

Marginal zone B cells (MZB) participate in the early immune response to several pathogens. In this study, we show that in μMT mice infected with Leishmania donovani, CD8 T cells displayed a greater cytotoxic potential and generated more effector memory cells compared with infected wild type mice. The frequency of parasite-specific, IFN-γ(+) CD4 T cells was also increased in μMT mice. B cells were able to capture parasites, which was associated with upregulation of surface IgM and MyD88-dependent IL-10 production. Moreover, MZB presented parasite Ags to CD4 T cells in vitro. Depletion of MZB also enhanced T cell responses and led to a decrease in the parasite burden but did not alter the generation of effector memory T cells. Thus, MZB appear to suppress protective T cell responses during the early stages of L. donovani infection.     

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F₁ or (A × C57BL/6) F₁ mice were injected with 10⁷ A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F₁ hybrid spleen cells) into corresponding unirradiated F₁ hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F₁ mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F₁ hosts not containing the antigens against which activation had been directed.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

Monoclonal helper T cells induce B cell responses to T-independent antigens: antigen-specific T cells are directly stimulated by activated B cells in the absence of antigen

Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.     

Corticosterone 21-acetate in vivo induces acute stress in chicken thymus: cell proliferation, apoptosis and cytokine responses

In vivo effects of acute stress induced by corticosterone 21-acetate in male Gallus domesticus thymus are studied and the steroid actions are evaluated in terms of cell proliferation, apoptosis and cytokine response in 10- and 21-day-old chickens. Steroid treatment induced thymocyte apoptosis and cell death decreased in the cortical-medullar direction and was more evident in younger animals. 24 h after treatment, the observed effect was reversed. The mitotic activity and thymic cells containing cytokine-like molecules were also affected. Indeed, the acute stress stimulated cytokine immunoreactivity to anti-IL-1alpha, IL-6 and TNF-alpha antibodies both in epithelial cells and interdigitating cells located in medullar and cortical-medullar regions. The increased cytokine expression observed after 12 h was maintained after 24 h. The comparison between 10- and 21-day-old chickens showed a lower number of cells containing cytokine-like molecules in younger specimens. The present findings suggest that cytokines activated by acute stress in vivo could contribute to restoring immunological homeostasis and influence thymic glucocorticoid-mediated functions.     

Enhancement and suppression of immunoglobulin G-producing B cells in the presence of immune T cells.

Mice were immunized one to three times with sheep red blood cells. Four to seven days after the last immunization, the spleens were removed and the cells were cultured in vitro in the absence of antigen. Removal of most T cells by anti-θ serum treatment prior to culture could increase the number of IgG-producing B cells without affecting the number of specific or nonspecific IgM-producing B cells detected after 2 days of culture. Addition of graded numbers of immune cells to pure immune B cells enhanced the number of IgG-producing B cells, whereas addition or higher number of immune cells caused suppression. Since removal of T cells could also enhance the proliferation of IgG-producing B cells induced by lipopolysaccharide (LPS), a polyclonal B-cell activator, it is suggested that the suppressive effects of high numbers of immune T cells are exerted directly on the B cells.     

Antigen presentation by splenic B cells: resting B cells are ineffective, whereas activated B cells are effective accessory cells for T cell responses

In this study, we have investigated the ability of splenic B cells to act as antigen-presenting cells. Previous data had established that lipopolysaccharide (LPS)-activated B cells were effective antigen-presenting cells; however, the relative capacity of resting B cells to carry out this function remains controversial. Splenic B cells from naive BALB/c mice were depleted of macrophages, dendritic cells, and T cells, and were fractionated on the basis of cell density by using Percoll gradient centrifugation. Fractions were collected from the 50/60, 60/65, and 65/72% interfaces and from greater than 72% (pellet). Cytofluorograph analysis of the fractionated B cells showed that the two lower density fractions (50/60 and 60/65) contained a number of cells which, by cell size determination, appeared to be activated B cells, whereas the two higher density fractions (65/72 and greater than 72) appeared to contain predominantly small resting B cells contaminated by many fewer activated B cells. Functionally, the capacity of fractionated B cells to act as accessory cells for a concanavalin A response or present the antigens chicken ovalbumin (OVA) or OVA-tryptic digest gave similar results, which indicated a striking hierarchy of accessory cell function in the different Percoll fractions. When normalized to the most active low-density fraction (50/60%), the activity of the other fractions were: 60/65 = 78%; 65/72 = 25%; and greater than 72 = 4%. The differences in the functional capacity between the various Percoll fractions did not appear to be due to differences in Ia expression. Although the expression of Ia varied approximately 12-fold within any one fraction, there was little difference in the mean amount of Ia on cells obtained from the various fractions. Kinetic studies showed that activation of B cells with LPS and dextran sulfate resulted in the expression of two stages of functional development. The first stage was an increased efficiency of accessory cell function that was abrogated by irradiation with 4000 rad followed by a second stage, which was characterized by the acquisition of resistance to treatment with 4000 rad. When nonfractionated B cells that had been stimulated with LPS and DexSO4 were sorted on the basis of cell size into a small B cell fraction and a large B cell fraction, only the large B cells were able to present antigen. Taken together, these data suggest that much of the accessory cell function associated with splenic B cells can be accounted for by the relatively small percentage of activated B cells present in the spleen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cognate T and B cell interaction and association of follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. T cells can play critical roles in inducing B cell mediated immunity. As the mechanism of T cell dependent B cell maturation is not well established, we hypothesized that a specific population of T (follicular helper T, Tfh) cells, are involved in B cell maturation following cholera. We found flowcytometrically that V. cholerae infection induces significant increases in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to V. cholerae membrane preparation (MP) and V. cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of co-cultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype colorimetrically. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in gut mucosa of biopsied samples, at the single cell level if feasible.