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Age-related thymic atrophy plays a significant role in delayed immune reconstitution in older recipients after hemopoietic stem cell transplantations (HSCT). Sex steroid ablation has been shown to reverse thymic atrophy and previously we have shown that in syngeneic and allogeneic models of HSCT, sex steroid ablation enhances immune reconstitution. Donor-derived HSC numbers, as well as precursor T and B cells are increased in castrated mice following HSCT. These primary changes lead to an increase in both T and B cells in the periphery. The current study examined both the molecular mechanisms behind this enhanced reconstitution and the function of the lymphocytes produced. Bone marrow (BM) and thymic stromal cell (TSCs) populations were analysed using RT-PCR and were tested for the production of growth factors previously implicated in immune reconstitution. Functional studies including proliferation and cytotoxicity assays and intracellular cytokine production showed that on a per cell basis, there was no difference between the T cells from castrated and sham-castrated mice, following allogeneic HSCT. In vivo immune function was assessed using a delayed type hypersensitivity assay. Six weeks after HSCT the DTH response was enhanced in the castrated mice compared to sham-castrated controls. The combination of a) the increased number of donor-derived lymphocytes and b) intact T cell function, result in an overall increase in immune response (as determined by DTH) in castrated recipients of allogeneic HSCT. This enhanced function may be related to changes seen in growth factor production in the thymus and bone marrow.

Different procedures for enrichment of peripheral blood leukocytes (PBL) from whole chicken blood have been compared in terms of the percentage yield of lymphocytes and thrombocytes. The yield of thrombocytes was low in the buffy coat cells of heparinised blood, but high in PBL enriched from heparinised blood over a Fiooll-Paque density gradient and in PBL prepared from citrated blood by either technique. The fluorescence activated cell sorter (FACS) has been used to separate thrombocytes from lymphocytes in PBL by sorting of the negative cells after staining with specific anti-T and anti-B cell sera (or anti-immunoglobulin). The sorted cells were more than 99% viable, more than 98% thrombocytes by antigenic and morphological criteria and did not respond invitro to T and B cell mitogens. Absorption studies on a turkey anti-bursa cell serum (ABST) which is cross-reactive for B cells and thrombocytes (8) indicated that the cross-reactivity is attributable to two (or more) antibody specificities in the serum rather than to one antibody directed at shared determinants on the two cell types.

Specific antisera reacting with B-L (Ia-like) antigens were prepared by reciprocal immunization of animals from the congeneic lines CB and CC. The resulting antisera were tested either in direct or indirect immunofluorescence tests and stained 10–16% of peripheral blood cells (PBL). Of the B-L+ cells, 90% were B cells and 8% were T cells. After invitro stimulation of PBL with Con A, 58% were B-L+ and 91% of these were T cells. B and T cells were defined by means of rabbit antisera raised against bursa and thymus cells made specific by absorption with the relevant tissues. Antigens determined by anti-B-L antisera, rabbit anti-bursa (ABS) and rabbit anti-thymus (ATS) sera showed an independent distribution on the membrane of PBL. The tissue distribution of B-L+ cells, defined by means of allo-antisera and monoclonal antibodies, was studied by direct and indirect immunofluorescence on sections of skin, liver, kidney and brain. In all organs, in addition to B cells and a small number of, presumably activated, T cells, macrophages and dendritic cells were positive. Notably, glia cells in the brain were also shown to express B-L antigen.

Surface antigens on chicken thymus and bursa cells were analyzed by immunoprecipitation using polyclonal and monoclonal antisera raised against (and specific for) thymus (ATS) or bursa (ABS) cells, respectively. The antigens identified were compared with those governed by the B-F, B-L and B-G regions of the chicken major histocompatibility complex (B complex). Four proteins were precipitated from thymus cells by 2 polyclonal ATS: both antisera recognized molecules of apparent molecular mass of 172–182, 132–135, 75–76 kDa, and one antiserum in addition recognized a protein of 102 kDa. The 172–182 and 102-kDa peaks were still demonstrable under reducing conditions indicating that they are composed of a single polypeptide chain, the other 2 were lost under reducing conditions, therefore, must be composed of smaller subunits. Of the 2 monoclonal ATS tested, one identified a single protein of 186 kDa and the other a 135-kDa protein (in addition to 2 smaller molecules); whether these are the same as those precipitated by the polyclonal antisera remains to be determined as they behaved differently under reducing conditions. Proteins of 162 and 78–84 kDa were revealed by 2 polyclonal ABS under nonreducing conditions but the former may in one case be a polymer (it disappeared under reducing conditions) and in the other a single molecule. In addition molecules of 182 kDa were identified by one antiserum and of 84 and 60 kDa by the other under nonreducing conditions. Of the 4 monoclonal ABS only one identified a 200-kDa protein: molecules of 115–125, 90–100, 48–52 and 40–43 kDa were also precipitated, all of which were reduced to smaller molecules. With 2 specific anti-B-F alloantisera we were able to precipitate the “conventional” B-F antigen from red blood cell lysates of CB-strain chickens resolving into a 40-kDa peak and a light chain of about 12 kDa corresponding to β2 microglobulin. Precipitates from peripheral blood lymphocytes, bursa and thymus cells revealed an additional protein of 22 kDa. With 2 specific B-L alloantisera two peaks of 33 kDa and 31 kDa were obtained from peripheral blood lymphocytes. Using anti-B-G alloantisera a double band corresponding to 47 and 42 kDa was seen under reducing conditions. There is no evidence from these data to indicate that the polyclonal and monoclonal antibodies are directed towards major histocompatibility complex antigens.

The ontogenic development of the bursa-dependent lymphoid tissue in the chicken has been studied using rabbit antisera specific for B lymphocyte sub-populations and two elements of the bursal microenvironment. The antigens investigated were: the chicken B lymphocyte antigen ( CBLA ); mature B lymphocyte antigen ( CMBLA ); a foetal-associated antigen ( CFAA ) present on embryonic haemopoietic cells and adult bone marrow and bursa cells; immunoglobulin (Ig) and IgG; a bursa-specific cortical reticulin fibre antigen ( CBRFA ); a gut-associated mucin antigen ( CGAMA ) present on bursal medullary reticular epithelial (REp) cells. The development of suspected precursor cells was examined using a rabbit antiserum specific for foetal spleen cells. The major finding was the interrelationship between developing B cells and the bursal microenvironment. The first CBLA - and CFAA -positive cells were detected in the bursa at day 8 of incubation but their precise localization was difficult to assess. In 12-15 day embryos, both these cells were found predominantly in the tunica-propria in close proximity to cells bearing the reticulin fibre antigen CBRFA . This close association between CBLA -, CFAA - and CBRFA determinants represents the earliest stages of B cell differentiation and mimics that found in the adult bursa cortex. By day 18, the majority of bursa cells expressed CBLA and Ig and were localized in the developing medullary follicles, the REp cells of which were CGAMA -positive, demonstrating a very early interaction between intestinal tract contents, bursal REp cells and B cell maturation. Around hatching some bursa cells showed a marked increase in the membrane expression of CBLA and Ig, and the simultaneous expression of CMBLA and IgG. These cells were present in medullary follicles; CBRFA was present on cortical reticulin fibres which provided a supporting framework for the more immature CFAA -positive cells.

The thymic nurse cell (TNC) consists of an epithelial cell enclosing lymphoid elements and is found in enzymic digests of the thymus. Although these structures have been implicated in the normal intrathymic development of T lymphocytes, little is known about the in situ structure of this unusual cell complex. In this study, various dyes were introduced into the intact thymus and their differential permeability was used to demonstrate that the TNC exists as a sealed structure in situ. The lymphocytes within the TNC were shown to be isolated from the general thymic environment. Preliminary studies on these lymphocytes and the physiology of their active release from individual, micromanipulated TNC in microcultures are reported.

Three subsets of chicken peripheral T cells (T1, T2 and T3) have been identified in peripheral blood of adult chickens on the basis of fluorescence intensity after staining with certain xenogeneic anti-thymus cell sera (from turkeys and rabbits). They differentiate between 3-10 weeks of age in parallel with development of responsiveness to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Functional tests on the T subsets, sorted with a fluorescence-activated cell sorter, have shown that T2, 3 cells respond to Con A, PHA and PWM and are capable of eliciting a graft-vs.-host reaction (GvHR). In contrast, although T1 cells respond to Con A, they respond poorly to PHA and not at all to PWM or in GvHR. There was some indication of cooperation between T1 and T2,3 cells for the PHA response. Parallels between these chicken subsets and helper and suppressor/cytotoxic subsets in mammalian systems are discussed.

SummaryImmature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

Despite the simple structural features of the thymus which standard histological examination reveals, the very intricate pathways of T lymphocyte differentiation occuring in this organ (1) would predict that the stromal elements which regulate this process must be equally complex. Tissue culture of dispersed thymic stromal cells (2), ultrastructural studies (3) and the distribution of MHC antigens (4) provided the first indications that this was so. More recently, use has been made of M.Abs. raised against enriched preparations of thymic stromal cells to identify a limited number of specific regions of the thymus which may reflect different roles in T cell maturation (5-7). Using the chicken thymus as a model, we have been able to markedly extend these observations by finding numerous antigenically distinct regions within which were multiple populations of stromal cells and their products (8; Boyd et al. submitted). This extensive panel of M.Abs to chicken stromal antigens probably reflects the phylogenetic distance between chickens and the mice used for immunization. We were interested to see, however, whether modified immunogen preparations and immunization protocols would reveal if similar complexities exist in the mouse and human thymus. This paper summarizes the major findings of these studies which clearly indicate that this is so.

The thymus has two main cellular zones, clearly distinguishable histologically; the major outer zone, the cortex, and the smaller central zone, the medulla. Although this morphological subdivision has been known for a long time, we still don’t know the respective roles of these two thymic compartments in the generation of peripheral T cells [reviewed in 1,2]. Our ignorance in this area is surprising since we have a lot of information concerning the nature of the lymphoid and non-lymphoid cells in the two compartments. Thus, isolated thymic lymphocytes fall into two major categories: “mature” cells binding low levels of peanut agglutinin, and expressing relatively little Thy 1 but a lot of H-2 antigen, and “immature” cells which are PNAhi, Thy 1h1 and H-2low [reviewed in 3]. The mature cells are located mainly in the medulla and hence we will call them medullary-phenotype cells, while the major population (85%) of immature cells is located in the cortex and hence these are termed cortical-phenotype cells. We use the term cortical- and medullary-phenotype because a small number of cells of either phenotype could exist in the “wrong” region and could thus be, for example, of medullary phenotype but located in the cortex. We should stress, though, that most cells do occur in the “right” region, and exceptions (for which there is little hard evidence, see below) must be few in number [see ref. 3].

The complex intra-thymic processes culminating in the generation of fully functional, self tolerant, self MHC restricted T lymphocytes have proven difficult to decipher. They appear to involve extensive interactions between T cell progenitors and thymic stromal cells or their soluble products. A better understanding of these mechanisms, however, has been hampered through the inability to identify and isolate individual sub-populations from the extremely heterogeneous thymic stroma.

The B-L region of the chicken major histocompatibility complex (MHC), the so-called B -locus, corresponds to the murine H-2 I-region. Using alloantibodies and monoclonal antibodies to B-L we analyzed: (a) the tissue distribution of B-L + cells, (b) the function of B-L + cells, and (c) the possible role of B-L + cells in the development of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens. The tissue distribution of B-L + cells in peripheral blood and various lymphoid and nonlymphoid organs corresponds to what is known for mammals. In the bursa of Fabricius most lymphoid cells and the dendritic cells carry the B-L antigen; B-L + thymic nurse cells (TNC) first appear on day 17 of embryonic life; chickens possess dendritic B-L + cells in the skin resembling mammlian Langerhans cells; in addition we found that the microglia is unequivocally B-L + . B-L + peripheral blood lymphocytes (PBL) were separated with a fluorescence-activated cell sorter. Ten percent of unstimulated PBL and 60% of phytohemagglutinin (PHA) stimulated T-cell blasts are B-L + . In graft-vs-host (GvH) assays B-L − cells were identified as the effector cells. These cells respond to PHA and concanavalin A (Con A), but not to pokeweed mitogen (PWM). B-L + cells cannot be stimulated by Con A and PHA, but respond to PWM. They possess only a very low activity in GvH assays which can be inhibited by anti-T-cell sera. In OS chickens B-L + /non-B/, non-T and B-L + T (blasts?) cells are found in the “first line” of mononuclear cell infiltration in the thyroid glands. Most interesting, thyroid epithelial cells—which are normally B-L − —become B-L + in the neighbourhood of B-L + infiltrating mononuclear cells. This observation may be of significance for autoantigen presentation and perpetuation in autoimmune thyroiditis. Finally, OS thymuses contain significantly less TNC than normal controls.

The Obese strain (OS) of chickens was developed about 15 years ago by selective breeding for the phenotypic trait of hypothyroidism [1, 2]. The fact that the clinical symptoms are attributable to a spontaneously arising thyroiditis which is autoimmune in nature was first recognized 5 years later [3]. In subsequent years this avian model was established as the closest counterpart to human Hashimoto’s disease [4]; the data accumulated by 1974 have already been presented in a comprehensive review [5].

The avian bursa of Fabricius provides a unique model for the investigation of B lymphocytopoiesis since its involvement in this process is essential (Grossi et al., 1976; Eerola et al., 1982; Boyd et al., 1983). This organ contributes significantly to the clonal expansion of B cells, the majority of follicles being derived from 2-5 stem cells committed prior to proliferation within the bursa (Pink et al., 1985b; Ratcliffe et al., 1986). Close to hatching, these differentiated cells acquire the ability to migrate to peripheral tissues (Boyd and Ward, 1984). A common precursor to the follicular cortex and medulla has been described (Pink et al., 1985a) and it appears less differentiated lymphocytes occur in the cortex (Boyd and Ward, 1978). It is also established that the bursal epithelium is receptive to stem cell influx between 8 and 15 days of embryogenesis (Le Douarin, 1986).

There is evidence of a role for thymic reticulum cells and epithelium in mammalian T lymphocyte differentiation (1,2) and we have previously shown (3) that chicken thymic and bursal reticular epithelial (REp) cells are able to induce in vitro the appearance of T and B lymphocyte surface markers, respectively, on embryonic precursor cells. In addition, bursa-derived factor(s) have been shown to influence B cell development (4,5). Chicken thymus REp cells have specific characteristics (6,7) and we have demonstrated (8) the antigenic specificity of the bursal reticulin framework associated with the cortico-medullary junction and the cortex. Thus, considerable evidence points to the specificity of reticular-and epithelial-type cells of primary lymphoid organs and to a role of these cells in lymphocyte differentiation. With a view to characterizing and investigating the function of avian REp cells and associated structures xenoantisera have been produced which have enabled the detection of a number of antigens.

The functional of B-L (Ia-equivalent)-positive (B-L+) adn -negative (B-L-) chicken peripheral blood lymphocytes (PBL) was studied in vitro and in vivo. The PBL were first stained in direct immunofluorescence tests with a fluorescein isothiocyanate-labelled anti-B-L alloantiserum and then separated by means of a fluorescence-activated cell sorter. In agreement with our previous findings, B-L- cells showed functional properties of T lymphocytes, responding to concanavalin A and phytohaemagglutinin-P in vitro and inducing a graft-versus-host (GVH) reaction when injected into allogeneic embryos. Sorted B-L+ gave no responses in any of these assays. Neither B-L+ nor B-L- cells, when tested alone, responded significantly to pokeweed mitogen, but mixtures of the two restored the responsiveness to that of the original unsorted suspension. Of the B-L+ PBL, 10% were T cells, which may account for the low GVH reactivity given by this population.

Antiserum produced by immunizing rabbits with embryonic chicken spleen cells showed, after appropriate absorption with cell suspensions, specificity for fetal spleen cells in membrane immunofluoresoence tests. However, when the original antiserum was absorbed with tissue homogenates and tested on cryostat tissue sections, it reacted specifically with the reticular framework which separates the cortical and medullary lymphocytes in the follicles of the adult bursa of Fabricius. It is suggested that the reticulin may influence bursal lymphoid differentiation.

Thymic nurse cells, cortical epithelial cells enclosing 20-200 lymphocytes, were prepared from mouse thymus by enzyme digestion and repetitive sedimentation. Individual nurse cells were then isolated free of any exogenous thymocytes by micromanipulation, and the endogeneous thymocytes released from inside the nurse cells by a brief period of culture. The thymocytes from within individual nurse cells were tested, at the one cell/well level, for their capacity to proliferate in high cloning efficiency mitogen-stimulated limiting dilution cultures. The resultant clones were tested for their cytolytic capacity in a lectin-mediated isotype-release assay. Most intra-nurse cell thymocytes were unresponsive, like typical cortical thymocytes, but an average of 1/30, or around 2-6 lymphocytes/nurse cell, were able to proliferate in response to concanavalin A. The clones produced were of a relatively small size, similar to those characteristic of helper-lineage T cells. No cytolytic clones at all were obtained, despite stringent positive controls showing an efficient cytolytic response from known sources of cytolytic precursor cells. This finding disagrees with earlier studies on nurse cell lymphocytes, where there may have been a possibility of contamination with exogenous thymocytes. These results suggest either that the nurse cell represents a selective environment for helper-lineage T cell differentiation, or that further steps after the nurse cell stage are needed to produce mature cytolytic-lineage T cells.

The thymus has generally been considered as being solely involved in T cell maturation. In this study we have demonstrated that mouse thymic stroma can also support myelopoiesis. Bone marrow from mice treated with 5-fluorouracil was depleted of cells expressing Mac-1, CD4, and CD8 and incubated on lymphocyte-free monolayer cultures of adherent thymic stromal cells. After 7 days there was a marked increase in nonadherent cells, the majority of which were Mac-1+, FcR+, and HSA+. These proliferating bone marrow cells also expressed markers (MTS 17 and MTS 37) found on thymic stromal cells. Such cells were not found in thymic cultures alone, in bone marrow cultured alone, or on control adherent cell monolayers. Supernatants from the cultured thymic stroma, however, were able to induce these cell types in the bone marrow precursor population. Incubation of normal thymocytes with a monolayer of these in vitro cultivated Mac-1+, MTS 17+, MTS 37+ myeloid cells leads to selective phagocytosis of CD4+ CD8+ cells. Hence, this study demonstrates that the thymic adherent cells can induce myelopoiesis in bone marrow-derived precursor cells and provide a form of self-renewal for at least one population of thymic stromal cells. Furthermore, these induced cells are capable of selective phagocytosis of CD4+ CD8+ thymocytes and may provide one mechanism for the selective removal of such cells from the thymus.

The undecapeptide Substance P (SP) which is contained in primary afferent nerves, has been shown to affect certain components of the neurogenic inflammatory response including vasodilatation and plasma extravasation. Substance P also modulates the in vitro responses of lymphocytes and accessory cells. Rats treated neonatally with the sensory neurotoxin capsaicin, show a diminished neurogenic inflammatory response, but the effects of capsaicin treatment on cells of the immune system have not been previously documented. Using Sprague-Dawley rats at 6–12 weeks of age that had been pretreated neonatally with capsaicin, we examined the primary antibody response of popliteal lymph-node lymphocytes to antigenic stimulation in vivo with sheep red blood cells. It was observed that the number of cells secreting antigen-specific immunoglobulins was decreased in the capsaicin pretreated rat by more than 85%. This reduced response was reversed by a sub-cutaneous infusion of SP at the injection site immediately following antigenic stimulation. The results suggest that the decreased response in the capsaicin treated rats is secondary to the neurotoxic effects of capsaicin, and is most probably mediated by SP.

Pluripotential stem cells appear to require discrete microenvironmental factors to differentiate along a particular developmental pathway. In the chicken, the avian bursa of Fabricius provides a complex collection of cells and factors (1), which enable blood borne precursor cells to differentiate into functional B-lymphocytes (2). The exact nature of the stromal components affecting B-cell differentiation, however, remains poorly defined.

Natural cell mediated immunity is a well-defined system that may play a role in cancer defence. In contrast to specific T-cell mediated immunity, the mediators of natural immunity, Natural Killer (NK) cells, appear to be non-specific and their lytic activity is not restricted by major histocompatibility (MHC) determinants1.

University of California, Davis (UCD) line 200 chickens spontaneously develop a progressive fibrotic syndrome with features similar to those observed in human autoimmune connective tissue diseases, including fibrosis, vascular occlusion, and lymphocytic infiltration of the comb, skin, digits, and viscera. Beginning at 2 weeks post hatch, line 200 chickens develop intense lymphocytic infiltration of the comb and dorsal neck skin. To further characterize the nature of these cellular infiltrates, weekly serial skin biopsy specimens from line 200 and control birds were examined using hematoxylin and eosin staining and indirect immunofluorescence with a library of mouse anti-chicken monoclonal antibodies specific for lymphocyte markers. In situ staining performed on serial skin sections revealed the presence of large groups of T cells beginning at 2 weeks of age. Further characterization of these infiltrates demonstrated the presence of both T helper and T cytotoxic/suppressor cells with a mean +/- SD T4:T8 ratio of 1.44 +/- 0.29 by 4 weeks of age. As the lesions progressed, the infiltrates also contained distinct groups of B cells as characterized by MUI 36. In addition, the lesions were strongly positive for B-L (Ia) antigen, which was noted on B cells, monocytes/macrophages, activated T cells, and fibroblasts. The skin sections were negative for 2 different macrophage monoclonal antibodies at all time-points. Upon extraction from affected skin, 42.0 +/- 13.06% (mean +/- SD) of these cells were positive for B-L, 35.10 +/- 6.51% were T cells, and 31.25 +/- 3.14% were recognized by MUI 36. Although positive staining for IgG was not found in these extracted cells, 7% of the isolated cells were positive for surface IgM.

Any detailed study of T-cell development within the thymus requires, as a first step, the ability of distinguish the subpopulations of lymphoid cells which may be steps along a developmental pathway. About 95% of adult murine thymic lymphocytes can be assigned to one of four discrete subpopulations as summarized in Table 1. These major subpopulations, and many of their surface markers, have been recognized for some time, and the recent application of multiparameter flow cytometric analysis (Scollay and Shortman 1983) has merely served to emphasize how clear cut this four-way division can be. This subdivision is obtained as follows.

The chicken MHC (B complex) initially described by Briles as controlling blood antigens, is now known to be composed of at least three regions, L, F and G. Two of these, F and G, were described on the basis of recombinants found in a study of over 10,000 chickens. On the basis of biochemical, tissue distribution and functional analyses, F corresponds to the murine H-2 K/D regions. The G region is unique to the chicken since the antigenic product is expressed only on erythrocytes and their progenitors. L was identified by serological studies and corresponds to the H-2 I region; the L antigen is expressed predominantly on B lymphocytes, monocytes and 10% of T lymphocytes, and differences in the L region result in variations in immune responsiveness. A number of functional similarities exist between the chicken MHC and that of other species such as regulation of graft rejection, graft-versus-host reaction (GVHR) and mixed lymphocyte reactions (MLR), mitogenic and immune responsiveness and resistance to RNA and DNA virus infection. The chicken MHC also controls the severity of autoimmune disease, as exemplified by the spontaneous thyroiditis of Obese strain (OS) chickens. It differs from mammalian MHC's by having of lower crossing-over frequency and no apparent gene duplication.

The reactivity of chicken, human, rat and mouse lymphoid tissues with human serum containing smooth muscle antibody was studied by indirect immunofluorescence. In bursal follicles, strong fluorescence was observed predominantly in the lymphocytes and reticular epithelial cells of the medulla, the basement membrane and a row of undifferentiated epithelial cells at the corticomedullary junction. Reticular epithelial cells in lymphocyte-depleted bursas from cyclophosphamide-treated chickens and cultures of bursal and thymic reticular epithelial cells also reacted with smooth muscle antibody. The thymus of all species showed only medullary staining; lymph nodes, spleen and gut-associated lymphoid tissue showed extensive cell staining of both T and B lymphocyte dependent areas, with weak reactivity in germinal centres. Embryologically, the time of appearance of bursal and thymic lymphocyte staining coincided with the development of medullary lymphocytes. In organ imprints and cell smears of bursa, thymus, lymph node and spleen, and in cell smears of bone marrow and peripheral blood lymphocytes, immunofluorescent staining was restricted to cells in close contact with each other. Suspensions of dispersed viable lymphoid cells were negative. Specificity of the staining reaction was established by its prevention after neutralization: absorption of the serum with homogenates of smooth muscle or actin derived from the same source. It is concluded that an actin-like protein is present in mature B and T lymphocytes and in reticular epithelial cells and that its expression in lymphocytes appears dependent on cell-to-cell contact. The presence of the actin-like protein in mature lymphocytes may provide a means of lymphocyte motility in vivo.

The culture of freshly isolated lymphocytes for three to five days in vitro with a lymphokine preparation rich in interleukin 2 (IL-2), results in the generation of effector cells capable of lysing a variety of tumour cells1. This phenomenon has been described as lymphokine activated killing (LAK) and represents a lytic system distinct from that of natural killer (NK) cells2 and cytotoxic T lymphocytes3. Subsequent studies have revealed that LAK cells mainly arise from non-B, non-T lymphoid cell precursors and are capable of expanding for short times both in vivo and in vitro when stimulated by IL-24.

For the last twelve years data were accumulated by several groups that spontaneous autoimmune thyroiditis (SAT) which develops in the Obese strain (OS) of chickens depends on the presence of an intact B-dependent portion of the immune system (for review see refs. 1,2). Four types of effector mechanisms can be conceived as initiators of this disease and have been studied in detail: (a) complement binding thyroglobulin autoantibodies (Tg-AAb) either transferred from the mother hen via the egg yolk into the embryo and newly hatched chick or produced by the chick itself; (b) noncomplement binding IgG Tg-AAb triggering antibody-dependent cellular cytotoxicity (ADCC) by K-cells; (c) the occurrence of B-cells or mature plasma cells within the thyroid epithelial cell lining, a process called “periopolesis,” where formation of Tg-AAb has been demonstrated in situ which can again either bind complement or not and lead to a destruction or neighboring thyroid epithelial cells by mechanisms (a) or (b); (d) cytotoxic T-cells (Tc).

Poor immune recovery is characteristic of bone marrow transplantation and leads to high levels of morbidity and mortality. The primary underlying cause is a compromised thymic function, resulting from age-induced atrophy and further compounded by the damaging effects of cytoablative conditioning regimes on thymic epithelial cells (TEC). Several strategies have been proposed to enhance T cell reconstitution. Some, such as the use of single biological agents, are currently being tested in clinical trials. However, a more rational approach to immune restoration will be to leverage the evolving repertoire of new technologies. Specifically, the combined targeting of TEC, thymocytes and peripheral T cells, together with the bone marrow niches, promises a more strategic clinical therapeutic platform.

The nervous, endocrine, and immune systems interact to adapt to infection, inflammation, and tissue injury. Neural control is mediated in several ways, one of them being through the neuroendocrine regulation of the secretion of hypothalamic and pituitary hormones. The hormonal effects on the immune system range from the impact of steroidal hormones, which exhibit inhibitory effects over immune functions, to growth hormone, prolactin and neurohypophyseal hormones, known to stimulate and modulate humoral and cellular aspects of the immune system. This review will discuss the mechanisms behind the immunomodulatory role of the neuroendocrine system, including the critically important feedback loops required to maintain balance for these bidirectional interactions and alterations that occur with age.

The immune system undergoes dramatic changes with age—the thymus involutes, particularly from puberty, with the gradual loss of newly produced naive T cells resulting in a restricted T cell receptor repertoire, skewed towards memory cells. Coupled with a similar, though less dramatic age-linked decline in bone marrow function, this translates to a reduction in immune responsiveness and has important clinical implications particularly in immune reconstitution following cytoablation regimes for cancer treatment or following severe viral infections such as HIV. Given that long-term reconstitution of the immune system is dependent on the bi-directional interplay between primary lymphoid organ stromal cells and the progenitors whose downstream differentiation they direct, regeneration of the thymus is fundamental to developing new strategies for the clinical management of many major diseases of immunological origin. This review will discuss the impact of aging on primary lymphoid organ niches and current approaches for thymic regeneration and immune reconstitution.

This chapter examines how the thymus imposes tolerance and proposes novel ways through which it may be manipulated to achieve acceptance of stem cell transplants. Stem cell-based research is a highly complex, rapidly evolving technology that offers the ultimate horizon in clinical therapy—the physical replacement of damaged or missing cells and tissues. Although such “cell-based” therapies are of unquestionable potential importance, unless they are of autologous origin, any transplantation of such cells will meet the fate of immune rejection. This fundamentally involves an active thymus to generate T-cells and to purge the evolving pool of “self-reactive” cells. The transplantation of autologous stem cells could theoretically circumvent these problems. The crucial role of thymus in tolerance induction makes it an obvious target for the induction of transplantation tolerance. Although the establishment of donor–host hematopoietic chimerism can lead to lifelong tolerance to alloantigens, thymic involution in adults presents a technical problem to this approach. The ability to reproducibly direct the differentiation of cell lines towards immune stem cell lineages, in addition to therapeutic tissues, will allow testing of such strategies for the induction of immune tolerance to stem cell transplants

The thymus, a bilobate gland located in the upper anterior thorax in higher vertebrates, is the major site of T-lymphocyte production. This primary lymphoid organ composed of various nonlymphoid elements, referred to as the thymic stroma, governs the maturation and tolerance induction of haematopoietic precursors into immune-competent nonautoreactive T cells. Whilst naturally involuting with age, the thymic stromal compartment remains dynamic in nature throughout life and retains the potential to regenerate. Keywords: thymocyte; T lymphocyte; T-cell receptor; differentiation; thymus

Age-associated thymic involution is accompanied by decreased thymic output. This adversely affects general immune competence and T cell recovery following cytoreductive treatments such as chemotherapy. A causal link between increasing sex steroids and age-related thymic atrophy is well established. Although castration has been demonstrated to regenerate the atrophied thymus, little is known about how this is initiated or the kinetics of thymocyte regeneration. The present study shows that although castration impacts globally across thymocyte development in middle-aged mice, the regenerative effects are initiated in the immature triple-negative compartment and early T lineage progenitors (ETP). Specifically, there was a reduction in number of ETP with age, which was restored following castration. There was, however, no change in ETP reconstitution potential in ETP at this age or following castration. Furthermore, in a chemotherapy-induced model of thymic involution, we demonstrate castration enhances intrathymic proliferation and promotes differentiation through the triple-negative program. Clinically, reversible sex steroid ablation is achieved hormonally, and thus presents a means of ameliorating immune inadequacies, for example, following chemotherapy for bone marrow transplantation. By improving our understanding of the kinetics of thymic recovery, this study will allow more appropriate timing of therapy to achieve maximal reconstitution, especially in the elderly.

A major underlying cause for aging of the immune system is the structural and functional atrophy of the thymus, and associated decline in T cell genesis. This loss of naive T cells reduces adaptive immunity to new stimuli and precipitates a peripheral bias to memory cells against prior antigens. Whilst multiple mechanisms may contribute to this process, the temporal alliance of thymic decline with puberty has implicated a causative role for sex steroids. Accordingly ablation of sex steroids induces profound thymic rejuvenation. Although the thymus retains some, albeit highly limited, function in healthy adults, this is insufficient for resurrecting the T cell pool following cytoablative treatments such as chemo- and radiation-therapy and AIDS. Increased risk of opportunistic infections and cancer relapse or appearance, are a direct consequence. Temporary sex steroid ablation may thus provide a clinically effective means to regenerate the thymus and immune system in immunodeficiency states.

Introduction Mesenchymal stem cells (MSCs) have been identified as a viable treatment for inflammatory bowel disease (IBD). MSCs derived from bone marrow (BM-MSCs) have predominated in experimental models whereas the majority of clinical trials have used MSCs derived from adipose tissue (AT-MSCs), thus there is little consensus on the optimal tissue source. The therapeutic efficacies of these MSCs are yet to be compared in context of the underlying dysfunction of the enteric nervous system innervating the gastrointestinal tract concomitant with IBD. This study aims to characterise the in vitro properties of MSCs and compare their in vivo therapeutic potential for the treatment of enteric neuropathy associated with intestinal inflammation. Methods BM-MSCs and AT-MSCs were validated and characterised in vitro. In in vivo experiments, guinea-pigs received either 2,4,6-trinitrobenzene-sulfonate acid (TNBS) for the induction of colitis or sham treatment by enema. MSCs were administered at a dose of 1x106 cells via enema 3 hours after the induction of colitis. Colon tissues were collected 24 and 72 hours after TNBS administration to assess the level of inflammation and damage to the ENS. MSC migration to the myenteric plexus in vivo was elucidated by immunohistochemistry and in vitro using a modified Boyden chamber assay. Results Cells exhibited multipotency and a typical surface immunophenotype for validation as bona fide MSCs. In vitro characterisation revealed distinct differences in growth kinetics, clonogenicity and cell morphology between MSC types. In vivo, BM-MSCs were comparatively more effective than AT-MSCs in attenuating leukocyte infiltration and neuronal loss in the myenteric plexus. MSCs from both sources equally ameliorated body weight loss, gross morphological damage to the colon, changes in the neurochemical coding of neuronal subpopulations and the reduction in density of extrinsic and intrinsic nerve fibres innervating the colon. MSCs from both sources migrated to the myenteric plexus in in vivo colitis and in an in vitro assay. Conclusions These data from in vitro experiments suggest that AT-MSCs are ideal for cellular expansion. However, BM-MSCs were more therapeutic in the treatment of enteric neuropathy and plexitis. These characteristics should be considered when deciding on the MSC tissue source.

Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT) effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.

Summary Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration.

Background The use of mesenchymal stem cells (MSCs) to treat inflammatory bowel disease (IBD) is of great interest because of their immunomodulatory properties. Damage to the enteric nervous system (ENS) is implicated in IBD pathophysiology and disease progression. The most commonly used model to study inflammation-induced changes to the ENS is 2,4,6-trinitrobenzene-sulfonate acid (TNBS)-induced colitis in guinea pigs; however, no studies using guinea pig MSCs in colitis have been performed. This study aims to isolate and characterise guinea pig MSCs and then test their therapeutic potential for the treatment of enteric neuropathy associated with intestinal inflammation. Methods MSCs from guinea pig bone marrow and adipose tissue were isolated and characterised in vitro. In in vivo experiments, guinea pigs received either TNBS for the induction of colitis or sham treatment by enema. MSCs were administered at a dose of 1 × 106 cells via enema 3 h after the induction of colitis. Colon tissues were collected 24 and 72 h after TNBS administration to assess the level of inflammation and damage to the ENS. The secretion of transforming growth factor-β1 (TGF-β1) was analysed in MSC conditioned medium by flow cytometry. Results Cells isolated from both sources were adherent to plastic, multipotent and expressed some human MSC surface markers. In vitro characterisation revealed distinct differences in growth kinetics, clonogenicity and cell morphology between MSC types. In an in vivo model of TNBS-induced colitis, guinea pig bone marrow MSCs were comparatively more efficacious than adipose tissue MSCs in attenuating weight loss, colonic tissue damage and leukocyte infiltration into the mucosa and myenteric plexus. MSCs from both sources were equally neuroprotective in the amelioration of enteric neuronal loss and changes to the neurochemical coding of neuronal subpopulations. MSCs from both sources secreted TGF-β1 which exerted neuroprotective effects in vitro. Conclusions This study is the first evaluating the functional capacity of guinea pig bone marrow and adipose tissue-derived MSCs and providing evidence of their neuroprotective value in an animal model of colitis. In vitro characteristics of MSCs cannot be extrapolated to their therapeutic efficacy. TGF-β1 released by both types of MSCs might have contributed to the attenuation of enteric neuropathy associated with colitis.

Little is known about the molecular mechanisms and transcriptional regulation that govern T cell selection processes and the differentiation of CD4+ and CD8+ T cells. Mice lacking the interferon regulatory transcription factor-1 (IRF-1) have reduced numbers of mature CD8+ cells within the thymus and peripheral lymphatic organs. Here we show that positive and negative T cell selection of two MHC class I-restricted TCR alphabeta transgenes, H-Y and P14, are impaired in IRF-1-/- mice. The absence of IRF-1 resulted in decreased expression of LMP2, TAP1, and MHC class I on thymic stromal cells. Despite decreased MHC class I expression on IRF-1-/- thymic stromal cells, the defect in CD8+ T cells development did not reside in the thymic environment, and IRF-1-/- stromal cells can fully support development of CD8+ thymocytes in in vivo bone marrow chimeras and in vitro reaggregation cultures. Moreover, IRF-1-/- thymocytes displayed impaired TCR-mediated signal transduction, and the induction of negative selection in TCR Tg thymocytes from IRF-1-/- mice required a 1000-fold increase in selecting peptide. We also provide evidence that IRF-1 is mainly expressed in mature, but not immature, thymocytes and that expression of IRF-1 in immature thymocytes is induced after peptide-specific TCR activation. These results indicate that IRF-1 regulates gene expression in developing thymocytes required for lineage commitment and selection of CD8+ thymocytes.

The thymus undergoes age-related atrophy, coincident with increased circulating sex steroids from puberty. The impact of thymic atrophy is most profound in clinical conditions that cause a severe loss in peripheral T cells with the ability to regenerate adequate numbers of naive CD4+ T cells indirectly correlating with patient age. The present study demonstrates that androgen ablation results in the complete regeneration of the aged male mouse thymus, restoration of peripheral T cell phenotype and function and enhanced thymus regeneration following bone marrow transplantation. Importantly, this technique is also applicable to humans, with analysis of elderly males undergoing sex steroid ablation therapy for prostatic carcinoma, demonstrating an increase in circulating T cell numbers, particularly naive (TREC+) T cells. Collectively these studies represent a fundamentally new approach to treating immunodeficiency states in humans.

A recent paper in PNAS (Swijnenburg et al., 2008) reveals that human embryonic stem cells are not immune privileged. Potential therapeutic use of these cells will thus depend on the development of new strategies to counter foreign graft rejection by patient immune systems.