REGULATION OF AUTOSENSITIZATION : THE IMMUNE ACTIVATION AND SPECIFIC INHIBITION OF SELF-RECOGNIZING THYMUS-DERIVED LYMPHOCYTES

We studied the mechanisms underlying the natural tolerance of thymus-derived (T) lymphocytes for self-antigens. Lymphocytes from the thymus or lymph nodes of inbred rats were autosensitized in vitro against monolayers of autochthonous thymus reticulum cells or syngeneic fibroblasts. Receptors for self-antigens were detected by the specific adherence of normal lymphocytes to syngeneic cells. The achievement of active cell-mediated autosensitization was assayed by measuring the immunospecific lysis of syngeneic target cells in vitro, or graft-versus-host (GvH) reactions in vivo. The following observations were made using these systems. (a) A fraction of normal lymphocytes was found to have specific surface receptors that are able to recognize self-antigens which seem to be accessible in vivo. These potentially self-reactive lymphocytes were activated by incubation with syngeneic or autochthonous cells in vitro. Hence, the elimination of potentially self-reactive lymphocytes cannot be the only basis for natural self-tolerance. Therefore, the maintenance of self-tolerance in vivo appears to involve suppression of the immune reactivity of such self-tolerant lymphocytes. (b) We found that control of autosensitization depends upon the inhibition of the recognition of self-antigens. A GvH reaction in vivo could not be suppressed once recognition of self-antigens had occurred in vitro. Moreover, studies of the kinetics of antigen recognition indicated that several hours of incubation in vitro were needed for the inactivation of factors specifically inhibiting self-recognition. (c) We found that factors which inhibit self-recognition are present in fresh autologous serum. Treatment of the lymphocytes, but not syngeneic adsorbing cells, with autologous serum prevented recognition of syngeneic antigens. Allogeneic serum did not prevent self-recognition, and autologous serum did not inhibit the recognition of foreign antigens. These findings indicate that natural tolerance of T lymphocytes to self-antigens can be regulated by serum factors which act on the lymphocytes. The immunospecificity of the inhibitory effect suggests that these factors may be soluble self-antigens in a tolerogenic form

AUTOSENSITIZATION IN VITRO

Autosensitization of rat or mouse lymphoid cells against syngeneic fibroblast antigens was induced in cell culture. Rat lymphoid cells autosensitized by this method were able to produce immunospecific lysis of syngeneic target fibroblasts in vitro or GvH reactions in newborn rats. Autosensitized mouse spleen cells mediated similar GvH reactions when injected into newborn mice. The nature of the system used to induce immunity in vitro appears to argue against the possibility that lymphocytes capable of reacting against self-antigens could arise by mutation in cell culture. Hence, it is likely that cells potentially reactive against self-antigens preexisted in the lymphoid cell donors. The ability of autosensitized cells to mediate immune reactions in vivo suggests that the immunogenic self-antigens present on sensitizing fibroblasts also were accessible in the intact animals. Loss of natural self-tolerance in vitro, therefore, can be explained most simply by the existence of lymphocytes which are reversibly tolerant to self. Hence, ontogenic elimination of potentially self-reactive cells may not be the only basis for natural tolerance. Regulatory mechanisms, such as antigen excess, may have to function in vivo to prevent differentiation of self-tolerant lymphocytes. These regulatory mechanisms appear to be annulled in the cell-culture system. The present system thus may offer a new approach to studies of tolerance and regulation of cellular immunity

REJECTION OF TUMOR ALLOGRAFTS BY MOUSE SPLEEN CELLS SENSITIZED IN VITRO

This paper reports a model system of cellular immunity in which allosensitization of mouse spleen cells is induced in vitro. Allosensitization was achieved by culturing spleen cells upon monolayers of allogeneic fibroblasts. The ability of the spleen cells to inhibit the growth of tumor allografts in vivo served as a functional assay of sensitization. We found that unsensitized spleen cells or spleen cells sensitized against unrelated fibroblast antigens had no inhibitory effect on the growth of allogeneic fibrosarcoma cells when they were injected together into irradiated recipients. In contrast, spleen cells which were specifically allosensitized in vitro were found to be highly effective in inhibiting the growth of an equal number of allogeneic tumor cells. Several times more spleen cells from mice sensitized in vivo were required to produce a similar immune effect. This confirms the findings of previous studies which indicate that sensitization in cell culture can promote the selection of specifically sensitized lymphocytes. Preincubating sensitizing fibroblasts with allo-antisera blocked the allosensitization of spleen cells. This suggests that antibodies binding to fibroblasts may inhibit the induction of sensitization by competing with lymphocytes for antigenic sites. Mouse spleen cells which were able to recognize and reject tumor allografts in vivo were unable to cause lysis of target fibroblasts in vitro. Such fibroblasts, however, were susceptible to lysis by rat lymphoid cells sensitized by a similar in vitro method. These findings indicate that the conditions required for lymphocyte-mediated lysis of target cells may not be directly related to the processes of antigen recognition and allograft rejection in vivo

The immune-body cytokine network defines a social architecture of cell interactions

BACKGROUND: Three networks of intercellular communication can be associated with cytokine secretion; one limited to cells of the immune system (immune cells), one limited to parenchymal cells of organs and tissues (body cells), and one involving interactions between immune and body cells (immune-body interface). These cytokine connections determine the inflammatory response to injury and subsequent healing as well as the biologic consequences of the adaptive immune response to antigens. We informatically probed the cytokine database to uncover the underlying network architecture of the three networks. RESULTS: We now report that the three cytokine networks are among the densest of complex networks yet studied, and each features a characteristic profile of specific three-cell motifs. Some legitimate cytokine connections are shunned (anti-motifs). Certain immune cells can be paired by their input-output positions in a cytokine architecture tree of five tiers: macrophages (MΦ) and B cells (BC) comprise the first tier; the second tier is formed by T helper 1 (Th1) and T helper 2 (Th2) cells; the third tier includes dendritic cells (DC), mast cells (MAST), Natural Killer T cells (NK-T) and others; the fourth tier is formed by neutrophils (NEUT) and Natural Killer cells (NK); and the Cytotoxic T cell (CTL) stand alone as a fifth tier. The three-cell cytokine motif architecture of immune system cells places the immune system in a super-family that includes social networks and the World Wide Web. Body cells are less clearly stratified, although cells involved in wound healing and angiogenesis are most highly interconnected with immune cells. CONCLUSION: Cytokine network architecture creates an innate cell-communication platform that organizes the biologic outcome of antigen recognition and inflammation. Informatics sheds new light on immune-body systems organization. REVIEWERS: This article was reviewed by Neil Greenspan, Matthias von Herrath and Anne Cooke

GLUCOCORTICOIDS AND CELLULAR IMMUNITY IN VITRO : FACILITATION OF THE SENSITIZATION PHASE AND INHIBITION OF THE EFFECTOR PHASE OF A LYMPHOCYTE ANTI-FIBROBLAST REACTION

We studied the influence of glucocorticoids on the sensitization phase as well as on the cytolytic effector phase of an in vitro lymphocyte-mediated immune reaction. Lymphocytes obtained from the spleens or lymph nodes of unimmunized inbred rats were sensitized against foreign rat or mouse embryonic fibroblasts in cell culture. The capacity of the sensitized lymphocytes to produce a cytolytic effect was tested by transferring them to target fibroblast cultures. Injury to target fibroblasts was measured by release of radioactive 51Cr from previously labeled fibroblasts or by direct count of viable fibroblasts after incubation with sensitized lymphocytes. Various concentrations of water-soluble hydrocortisone or prednisolone were added to cell cultures during the 5 day sensitization phase and/or during the subsequent cytolytic effector phase and the influence of these hormones on the number and cytolytic capacity of the lymphocytes was measured. During the sensitization phase, the presence of glucocorticoid hormones, at concentrations of about 1 µg/ml, led to a profound decrease in the total number of recoverable lymphocytes. However, the per cent of large transformed lymphocytes was much greater in these treated cultures. The antigen-specific cytolytic capacity per cell of the glucocorticoid-treated lymphocytes, after the hormone was removed, was several times greater than that of lymphocytes sensitized in the absence of added hormones. Glucocorticoids influenced the effector phase of the reaction by inhibiting lymphocyte-mediated injury to target fibroblasts. The hormones, at concentrations of about 1 µg/ml, inhibited the cytolytic effect by about 50% without reducing the viability of the sensitized lymphocytes. Dose-dependent toxicity to lymphocytes and increasing inhibition of cytolytic effect appeared at higher concentrations of hormones. Thus, hydrocortisone and prednisolone, at concentrations of about 1 µg/ml, did not suppress the induction of sensitization, a process which they seem to facilitate in vitro. However, similar concentrations of these hormones appear to inhibit the cytolytic effector mechanism of sensitized lymphocytes. These findings may be relevant to the use of glucocorticoids as immunosuppressive agents in vivo

Emergent Dynamics of Thymocyte Development and Lineage Determination

Experiments have generated a plethora of data about the genes, molecules, and cells involved in thymocyte development. Here, we use a computer-driven simulation that uses data about thymocyte development to generate an integrated dynamic representation—a novel technology we have termed reactive animation (RA). RA reveals emergent properties in complex dynamic biological systems. We apply RA to thymocyte development by reproducing and extending the effects of known gene knockouts: CXCR4 and CCR9. RA simulation revealed a previously unidentified role of thymocyte competition for major histocompatability complex presentation. We now report that such competition is required for normal anatomical compartmentalization, can influence the rate of thymocyte velocities within chemokine gradients, and can account for the disproportion between single-positive CD4 and CD8 lineages developing from double-positive precursors

The Immune System Computes the State of the Body: Crowd Wisdom, Machine Learning, and Immune Cell Reference Repertoires Help Manage Inflammation

Here, we outline an overview of the mammalian immune system that updates and extends the classical clonal selection paradigm. Rather than focusing on strict self-not-self discrimination, we propose that the system orchestrates variable inflammatory responses that maintain the body and its symbiosis with the microbiome while eliminating the threat from pathogenic infectious agents and from tumors. The paper makes four points: The immune system classifies healthy and pathologic states of the body—including both self and foreign elements—by deploying individual lymphocytes as cellular computing machines; immune cells transform input signals from the body into an output of specific immune reactions.Rather than independent clonal responses, groups of individually activated immune-system cells co-react in lymphoid organs to make collective decisions through a type of self-organizing swarm intelligence or crowd wisdom.Collective choices by swarms of immune cells, like those of schools of fish, are modified by relatively small numbers of individual regulators responding to shifting conditions—such collective inflammatory responses are dynamically responsive.Self-reactive autoantibody and T-cell receptor (TCR) repertoires shared by healthy individuals function in a biological version of experience-based supervised machine learning. Immune system decisions are primed by formative experience with training sets of self-antigens encountered during lymphocyte development; these initially trained T cell and B cell repertoires form a Wellness Profile that then guides immune responses to test sets of antigens encountered later. This experience-based machine learning strategy is analogous to that deployed by supervised machine-learning algorithms.We propose experiments to test these ideas. This overview of the immune system bears clinical implications for monitoring wellness and for treating autoimmune disease, cancer, and allograft reactions

Mouse Dendritic Cells Pulsed with Capsular Polysaccharide Induce Resistance to Lethal Pneumococcal Challenge: Roles of T Cells and B Cells

Mice are exceedingly sensitive to intra-peritoneal (IP) challenge with some virulent pneumococci (LD50 = 1 bacterium). To investigate how peripheral contact with bacterial capsular polysaccharide (PS) antigen can induce resistance, we pulsed bone marrow dendritic cells (BMDC) of C57BL/6 mice with type 4 or type 3 PS, injected the BMDC intra-foot pad (IFP) and challenged the mice IP with supra-lethal doses of pneumococci. We examined the responses of T cells and B cells in the draining popliteal lymph node and measured the effects on the bacteria in the peritoneum and blood. We now report that: 1) The PS co-localized with MHC molecules on the BMDC surface; 2) PS-specific T and B cell proliferation and IFNγ secretion was detected in the draining popliteal lymph nodes on day 4; 3) Type-specific resistance to lethal IP challenge was manifested only after day 5; 4) Type-specific IgM and IgG antibodies were detected in the sera of only some of the mice, but B cells were essential for resistance; 5) Control mice vaccinated with a single injection of soluble PS did not develop a response in the draining popliteal lymph node and were not protected; 6) Mice injected with unpulsed BMDC also did not resist challenge: In unprotected mice, pneumococci entered the blood shortly after IP inoculation and multiplied exponentially in both blood and peritoneum killing the mice within 20 hours. Mice vaccinated with PS-pulsed BMDC trapped the bacteria in the peritoneum. The trapped bacteria proliferated exponentially IP, but died suddenly at 18–20 hours. Thus, a single injection of PS antigen associated with intact BMDC is a more effective vaccine than the soluble PS alone. This model system provides a platform for studying novel aspects of PS-targeted vaccination