G. Jeanette Thorbecke

CD4+ T helper cells engineered to produce latent TGF-β1 reverse allergen-induced airway hyperreactivity and inflammation

T helper 2 (Th2) cells play a critical role in the pathogenesis of asthma, but the precise immunological mechanisms that inhibit Th2 cell function in vivo are not well understood. Using gene therapy, we demonstrated that ovalbumin-specific (OVA-specific) Th cells engineered to express latent TGF-beta abolished airway hyperreactivity and airway inflammation induced by OVA-specific Th2 effector cells in SCID and BALB/c mice. These effects correlated with increased concentrations of active TGF-beta in the bronchoalveolar lavage (BAL) fluid, demonstrating that latent TGF-beta was activated in the inflammatory environment. In contrast, OVA-specific Th1 cells failed to inhibit airway hyperreactivity and inflammation in this system. The inhibitory effect of TGF-beta-secreting Th cells was antigen-specific and was reversed by neutralization of TGF-beta. Our results demonstrate that T cells secreting TGF-beta in the respiratory mucosa can indeed regulate Th2-induced airway hyperreactivity and inflammation and suggest that TGF-beta-producing T cells play an important regulatory role in asthma.

Role of IgD and Tδ Cells in the Regulation of the Humoral Immune Response

Since its discovery by Rowe & Fahey (1965), IgD has not been assigned a clearly defmed function. In fact, the role of IgD in the immune response is generally described in terms of what it does not do, rather than what it does. Thus, it is not secreted following antigenic or mitogenic stimulation of IgD+ B cells, does not appear to neutrahze antigen and does not fix complement. Taken together with the well established fact that IgD is present on the surface of the majority of B lymphocytes, these negative functional criteria have been used to support the view that IgD is primarily a cell-surface antigen receptor (Pemis 1971). The probable importance of IgD in the humoral immune system is underscored by the fact that the forces of evolution have ensured the conservation of the delta (S) heavy chain gene in phylogenetically divergent species (Leslie & Martin 1978). Indeed, it has been suggested that the delta heavy chain gene system originated early in evolution and branched from the alpha chain shortly after divergence of tbe gene systems for alpha and mu (Lin & Putnam 1981, Putnam et al. 1981).

Growth of SJL/J-derived transplantable reticulum cell sarcoma as related to its ability to induce T-cell proliferation in the host: III. Studies on thymectomized and congenitally athymic SJL mice

Abstract When SJL mice are irradiated and reconstituted with syngeneic bone marrow (XBM) they support growth of transplantable reticulum cell sarcoma to approximately 60% of that in normal mice. The ability to support RCS growth gradually improves with time after irradiation and reaches 90% of normal by 8–12 weeks. However, if the mice are thymectomized 4 weeks prior to treatment (Tx-XBM) they initially show 50% which increases to only 65% of growth in normal mice after 12 weeks. The ability of lymphoid cells from these mice to proliferate in vitro in response to irradiated RCS cells is normal 4 weeks after treatment in XBM, but remains in vivo possibly via their tendency to proliferate upon exposure to RCS.

Production of auto-anti-idiotypic antibody during the normal immune response: IX. Characteristics of the auto-anti-idiotype antibody and its production

Using hapten-reversible inhibition of plaque formation as an assay for auto-anti-idiotype antibody (anti-Id) and as a means for following idiotype (Id) expression, we have obtained evidence that following immunization with trinitrophenyl (TNP) conjugates (a) there are differences in Id expression in the anti-TNP antibody response to different TNP conjugates although there is some overlap; (b) different strains, although showing some differences in Id expression, tend to produce cross-reactive Ids, thus no obvious allotype linked inheritance of Id expression is observed in this heterogeneous immune response; (c) the auto-anti-Id produced following immunization with TNP-Brucella abortus or TNP-Ficoll tends to be of the IgG2a and IgG2b isotypes.

Proliferative and cytotoxic immune functions in aging mice. III. Exogenous interleukin-2 rich supernatant only partially restores alloreactivity in vitro

The ability of exogenous interleukin-2 (IL-2) rich supernatant to restore the defective T cell mediated immune functions of spleen cells from aged C57BL/6 mice was analyzed. Addition of IL-2 rich supernatant to allogeneic mixed lymphocyte cultures (MLC) resulted in an increase in the proliferative response of spleen cells from both young and old mice. The MLC response of cells from old mice was, however, not restored to the level of proliferation seen with splenocytes from young animals. In studying the generation of specific T cell suppressor function, it was found that IL-2 rich supernatant enhanced this function only for spleen cells from those aged animals which demonstrated a defective response in its absence. The response of these mice was thereby restored to the normal level. The response of cells from young control animals and aged mice with normal suppressor activity was not affected by the addition of IL-2 rich supernatant. We conclude that decreased IL-2 production constitutes a functionally important aspect, but is by no means the only defect in the immune response of aged mice. The results also suggest that responsiveness to IL-2 is less affected by age than lymphokine production.

Human T cell IgD receptors react with O-glycans on both human IgD and IgA1.

Previous studies on murine T cell IgD‐R have shown that these receptors recognize N‐glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD‐R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD‐coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, −2, −3, −4, IgA2, IgE and IgM fail to inhibit significantly at 20 μg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 μg and 10 μg/assay. Human IgA1 and IgD both contain Gal‐1 → 3‐GalNac‐rich O‐linked glycans, and on this basis are both bound to ricin and jacalin. The O‐linked glycans may therefore also represent the common moiety binding to IgD‐R. Disaccharides Gal‐1 → 3‐GalNac, and Gal‐1 → 4‐Glc at 10 μg/assay blocked IgD rosetting while Gal‐1 → 6‐Glc did not. We conclude that the human IgD‐R is a lectin, differing from the murine IgD‐R in that it has both IgA1 and IgD as ligands.

Migratory patterns of B lymphocytes. I. Fate of cells from central and peripheral lymphoid organs in the rabbit and its selective alteration by anti-immunoglobulin.

Abstract Homing properties of 3H-adenosine-labeled bone marrow (BM), appendix (App), and mesenteric (Mes) and popliteal (Popl) lymph node (LN) cells were studied in the rabbit. While BM cells 24–48 hr after transfer produced equivalent radioactivity (cpm/mg) in App, Mes, and Popl LN of recipients, App and Popl LN cells did not, showing a highly significant preference for their organ of origin. “Homing to antigen” was shown if Popl LN cells were taken from rabbits immunized to the same antigen as was injected in one footpad of recipients 2 weeks earlier. Pretreatment of App cells with sheep anti-rabbit Ig abolished their preferential localization in App as judged in recipients killed 5 hr after transfer, but this was a transient effect and no longer demonstrable by 24 hr. Histological localization of labeled cells showed the corona of follicles in the center of dome-shaped areas of the App to represent “B-influx areas” after transfer of all cell types and this localization was blocked temporarily (5 hr) by prior incubation of App cells with anti-Ig. Thymus-dependent interfollicular areas showed labeled cells after LN cell transfer, and less after App cells, but none after BM. Emphasized in the discussion are (i) the possible effect of antigen on homing of B cells and (ii) the implications of the findings with respect to the appendix as a peripheral lymphoid organ.

Multiple suppressive effects of transforming growth factor β1 on the immune response in chickens

The immunosuppressive effect of human recombinant TGF-beta 1 on chicken immune responses in vitro was evaluated. TGF-beta 1 at 1-10 ng/ml reduced T cell proliferation in response to concanavalin A by 50-80% and B cell proliferation in response to LPS by greater than 90%. In contrast, when added to immune spleen cells, it reduced the secondary PFC response to sheep erythrocytes by less than 50%, particularly when added at the same time as antigen on Day 2 of incubation. When TGF-beta 1 was added during a 2-day incubation to nylon wool-nonadherent immune or normal spleen cells, it caused the maintenance and/or appearance of suppressor cells. These suppressor cells, in coculture with immune spleen cells, inhibited the secondary PFC response in vitro without any further exposure to TGF-beta 1. The phenotype of the cells giving rise to suppressor cells under the influence of TGF-beta 1 was CT8+, TCR2+(alpha,beta), CT4-, TCR1-(gamma,delta) cells. The results suggest that, in addition to direct suppressive effects on the proliferation of B cells and of some T cells, TGF-beta 1 may suppress immune responses by maintaining or by promoting the development of suppressor T cells.

Role of endogenously produced interleukin-6 as a second signal in murine thymocyte proliferation induced by multiple cytokines: Regulatory effects of transforming growth factor-β1☆

Previous studies have demonstrated that murine thymocytes proliferate in the presence of submitogenic concentrations of phytohemagglutinin-P (PHA-P) and various cytokines such as interleukin-1 (IL-1), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). We report that C3H/HeJ thymocytes stimulated with PHA-P and IL-1, IL-4, or TNF-alpha secrete significant levels of IL-6 as determined on B9 hybridoma cells. The possibility that thymocyte proliferation induced by these cytokines was mediated through IL-6 was investigated utilizing a neutralizing monoclonal antibody against murine IL-6, MP5 20F3.1. The results demonstrate that MP5 20F3.1 inhibited the proliferative response of thymocytes and B9 hybridoma cells to recombinant MuIL-6 (but not HuIL-6) and neutralized the endogenous IL-6 produced in the thymocyte cultures, but did not have any measurable effects on the proliferative responses induced by IL-1, IL-4, or TNF-alpha. Although the level of endogeneously produced IL-6 did not play a measurable role in the proliferative response induced by TNF-alpha, the addition of higher concentrations of IL-6 augmented the proliferation of murine thymocytes induced by rMu TNF-alpha. In addition, recombinant human transforming growth factor-beta 1 (rHu TGF-beta 1) significantly inhibited thymocyte proliferation induced by HuIL-1, rMuIL-4, rMuIL-6, and rMuTNF-alpha. The studies suggest that IL-1, IL-4, or TNF-alpha mediate a proliferative signal on murine thymocytes independent of IL-6 and that the proliferative signals provided by these cytokines as well as IL-6 are inhibitable by rHu TGF-beta 1.

Production of auto-anti-idiotypic antibody during the normal immune response. VI. Hapten augmentation of plaque formation and hapten-reversible inhibition of plaque formation as assays for anti-idiotype antibody

(1) Evidence has been presented that the detection of hapten-augmentable plaques indicates cells whose secretion of antibody had been blocked by the binding of auto-anti-id to cell surface idiotypes. Because of the dependence of the assay on the affinities of the various species for one another, the number of hapten-augmentable plaques detected should be regarded as a minimal estimate of the number of cells whose secretion of antibody is inhibited by auto-anti-id. For confirmation that hapten-augmentable PFC are due to auto-anti-id 2 principal controls are important: (a) incubation of the spleen cell population with hapten prior to plaquing should remove the hapten-augmentable PFC; (b) the dialyzed supernate from hapten incubated cells should inhibit plaque formation in a hapten-reversible manner. (2) Evidence has been presented that hapten-reversible inhibition of plaque formation can serve as an assay for anti-id. Apparent false positive assays can result from the presence of anti-hapten antibody or antigen-antibody complexes; however, these apparent false positives are rarely reversed by hapten. Removal of anti-hapten antibody, by passage over an antigen immunoadsorbent, will eliminate this source of false positives and the procedure is recommended. False negative results can arise from mismatching of the anti-ids in the sample to be assayed and the idiotypes of the target cells used in the assay. This can result from shifts in idiotype expression related to age and time after antigen injection. False negatives can also result from the presence of idiotype-anti-id complexes in the sample to be assayed. This source of false negatives can sometimes be eliminated by passage of the sample through an antigen immunoadsorbent.