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Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4(+)T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β. Moreover, small intestinal eosinophils isolated from IL-1Ra-deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra., (© 2016 Sugawara et al.)

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

In conditions of T lymphopenia, interleukin (IL) 7 levels rise and, via T cell receptor for antigen-self-major histocompatibility complex (MHC) interaction, induce residual naive T cells to proliferate. This pattern of lymphopenia-induced "homeostatic" proliferation is typically quite slow and causes a gradual increase in total T cell numbers and differentiation into cells with features of memory cells. In contrast, we describe a novel form of homeostatic proliferation that occurs when naive T cells encounter raised levels of IL-2 and IL-15 in vivo. In this situation, CD8(+) T cells undergo massive expansion and rapid differentiation into effector cells, thus closely resembling the T cell response to foreign antigens. However, the responses induced by IL-2/IL-15 are not seen in MHC-deficient hosts, implying that the responses are driven by self-ligands. Hence, homeostatic proliferation of naive T cells can be either slow or fast, with the quality of the response to self being dictated by the particular cytokine (IL-7 vs. IL-2/IL-15) concerned. The relevance of the data to the gradual transition of naive T cells into memory-phenotype (MP) cells with age is discussed.

Survival and intermittent proliferation of memory CD4(+) and CD8(+) T cells appear to be controlled by different homeostatic mechanisms. In particular, contact with interleukin (IL)-15 has a decisive influence on memory CD8(+) cells, but not memory CD4(+) cells. Past studies of memory CD4(+) cells have relied heavily on the use of naturally occurring memory phenotype (MP) cells as a surrogate for antigen (Ag)-specific memory cells. However, we show here that MP CD4(+) cells contain a prominent subset of rapidly proliferating major histocompatibility complex (MHC) II-dependent cells. In contrast, Ag-specific memory CD4 cells have a slow turnover rate and are MHC II independent. In irradiated hosts, these latter cells ignore IL-15 and expand in response to the elevated levels of IL-7 in the lymphopenic hosts. In contrast, in normal nonlymphopenic hosts where IL-7 levels are low, memory CD4 cells are heavily dependent on IL-15. Significantly, memory CD4(+) responsiveness to endogenous IL-15 reflects marked competition from other cells, especially CD8(+) and natural killer cells, and increases considerably after removal of these cells. Therefore, under normal physiological conditions, homeostasis of CD8(+) and CD4(+) memory cells is quite similar and involves IL-15 and IL-7.

Most memory phenotype (MP) CD44(hi) CD8(+) cells are resting interleukin (IL)-15-dependent cells characterized by high expression of the IL-2/IL-15 receptor beta (CD122). However, some MP CD8(+) cells have a CD122(lo) phenotype and are IL-15 independent. Here, evidence is presented that the CD122(lo) subset of MP CD8(+) cells is controlled largely by major histocompatibility complex (MHC) class I molecules. Many of these cells display surface markers typical of recently activated T cells (CD62L(lo), CD69(hi), CD43(hi), and CD127(lo)) and show a high rate of background proliferation. Cells with this phenotype are highly enriched in common gamma chain-deficient mice and absent from MHC-I(-/-) mice. Unlike CD122(hi) CD8(+) cells, CD122(lo) MP CD8(+) cells survive poorly after transfer to MHC-I(-/-) hosts and cease to proliferate. Although distinctly different from typical antigen-specific memory cells, CD122(lo) MP CD8(+) cells closely resemble the antigen-dependent memory CD8(+) cells found in chronic viral infections.

Mice that are deficient in suppressor of cytokine signaling-1 (SOCS-1) succumb to neonatal mortality that is associated with extensive cellular infiltration of many tissues. T cells seem to be necessary for disease, which can be alleviated largely by neutralizing interferon-gamma. Examining T cell receptor (TCR) specificity shows that even monospecific T cells can mediate disease in SOCS-1-deficient mice, although disease onset is substantially faster with a polyclonal T cell repertoire. A major phenotype of SOCS-1-/- mice is the accumulation of CD44(high)CD8+ peripheral T cells. We show that SOCS-1-deficient CD8, but not CD4, T cells proliferate when transferred into normal (T cell-sufficient) mice, and that this is dependent on two signals: interleukin (IL)-15 and self-ligands that are usually only capable of stimulating homeostatic expansion in T cell-deficient mice. Our findings reveal that SOCS-1 normally down-regulates the capacity of IL-15 to drive activation and proliferation of naive CD8 T cells receiving TCR survival signals from self-ligands. We show that such dysregulated proliferation impairs the deletion of a highly autoreactive subset of CD8 T cells, and increases their potential for autoimmunity. Therefore, impaired deletion of highly autoreactive CD8 T cells, together with uncontrolled activation of naive CD8 T cells by homeostatic survival ligands, may provide a basis for the T cell-mediated disease of SOCS-1-/- mice.

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The gammaC cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of gammaC cytokine-responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.

Cytokines, particularly those of the common gamma chain receptor family, provide extrinsic signals that regulate naive CD4 cell survival. Whether these cytokines are required for the maintenance of memory CD4 cells has not been rigorously assessed. In this paper, we examined the contribution of interleukin (IL) 7, a constitutively produced common gamma chain receptor cytokine, to the survival of resting T cell receptor transgenic memory CD4 cells that were generated in vivo. IL-7 mediated the survival and up-regulation of Bcl-2 by resting memory CD4 cells in vitro in the absence of proliferation. Memory CD4 cells persisted for extended periods upon adoptive transfer into intact or lymphopenic recipients, but not in IL-7- mice or in recipients that were rendered deficient in IL-7 by antibody blocking. Both central (CD62L+) and effector (CD62L-) memory phenotype CD4 cells required IL-7 for survival and, in vivo, memory cells were comparable to naive CD4 cells in this regard. Although the generation of primary effector cells from naive CD4 cells and their dissemination to nonlymphoid tissues were not affected by IL-7 deficiency, memory cells failed to subsequently develop in either the lymphoid or nonlymphoid compartments. The results demonstrate that IL-7 can have previously unrecognized roles in the maintenance of memory in the CD4 cell population and in the survival of CD4 cells with a capacity to become memory cells.

Previous work has shown that memory-phenotype CD44(hi) CD8(+) cells are controlled by a cytokine, interleukin (IL)-15. However, the dependency of CD44(hi) CD8(+) cells on IL-15 is partial rather than complete. Here, evidence is presented that CD44(hi) CD8(+) cells comprise a mixed population of IL-15-dependent and IL-15-independent cells. The major subset of CD122(hi) CD44(hi) CD8(+) cells is heavily dependent on IL-15 by three different parameters, namely (1) "bystander" proliferation induced via IFN-induced stimulation of the innate immune system, (2) normal "background" proliferation, and (3) T cell survival; IL-15 dependency is most extreme for the Ly49(+) subset of CD122(hi) CD44(hi) CD8(+) cells. In contrast to CD122(hi) cells, the CD122(lo) subset of CD44(hi) CD8(+) cells is IL-15 independent; likewise, being CD122(lo), CD44(hi) CD4(+) cells are IL-15 independent. Thus, subsets of memory-phenotype T cells differ radically in their sensitivity to IL-15.

The overall size and composition of the pool of naive and memory T cells are tightly regulated by homeostatic mechanisms. Recent work has shown that homeostasis of naive T cells is controlled by two factors, self-major histocompatibility complex (MHC)/peptide ligands and a cytokine, interleukin (IL)-7. In particular, contact with these two factors is required for naive CD4+ and CD8+ cells to undergo "homeostatic" proliferation, i.e., proliferation induced as a consequence of severe T cell depletion. In contrast to naive T cells, the factors that drive memory T cells to undergo homeostatic proliferation are poorly understood. To address this issue, purified memory phenotype CD4+ and CD8+ cells from normal mice were adoptively transferred into various gene-knockout mice rendered T cell-deficient by sublethal irradiation. Three findings are reported. First, unlike naive T cells, homeostatic proliferation of memory T cells is largely MHC independent. Second, memory CD8+ cells can utilize either IL-7 or IL-15 to undergo homeostatic proliferation; however, in the absence of both IL-7 and IL-15, homeostatic proliferation fails to occur. Third, unlike memory CD8+ cells, homeostatic proliferation of memory CD4+ cells is independent of IL-7 and IL-15 (also IL-4). Thus, the homeostatic proliferation mechanisms that control memory CD8+ cells and memory CD4+ cells are quite distinct.

Transgenic (TG) mice expressing a high copy number of interleukin (IL)-7 cDNA under the control of the major histocomaptability complex (MHC) class II promoter display a 10-20-fold increase in total T cell numbers. Here, we show that the increase in T cell numbers in IL-7 TG mice is most apparent at the level of memory phenotype CD44hi CD122hi CD8+ cells. Based on studies with T cell receptor (TCR) TG mice crossed to IL-7 TG mice, increased levels of IL-7 may provide costimulation for TCR recognition of self-MHC ligands and thus cause naive CD8+ cells to proliferate and differentiate into memory phenotype cells. In addition, a marked increase in CD44hi CD122hi CD8+ cells was found in IL-7 TG IL-15(-) mice. Since these cell are rare in normal IL-15(-) mice, the dependency of memory phenotype CD8+ cells on IL-15 can be overcome by overexpression of IL-7.

Examining the rate of in vivo T cell turnover (proliferation) in aged mice revealed a marked reduction in turnover at the level of memory-phenotype CD44(hi) CD8(+) cells relative to young mice. Based on adoptive transfer experiments, the reduced turnover of aged CD44(hi) CD8(+) cells reflected an inhibitory influence of the aged host environment. Aged CD44(hi) CD8(+) cells also showed poor in vivo responses to IL-15 and IL-15-inducing agents, but responded well to IL-15 in vitro. Two mechanisms could account for the reduced turnover of aged CD44(hi) CD8(+) cells in vivo. First, aging was associated with a prominent and selective increase in Bcl-2 expression in CD44(hi) CD8(+) cells. Hence, the reduced turnover of aged CD44(hi) CD8(+) cells may in part reflect the antiproliferative effect of enhanced Bcl-2 expression. Second, the impaired in vivo response of aged CD44(hi) CD8(+) cells to IL-15 correlated with increased serum levels of type I interferons (IFN-I) and was largely reversed by injection of anti-IFN-I antibody. Hence the selective reduction in the turnover of aged CD44(hi) CD8(+) cells in vivo may reflect the combined inhibitory effects of enhanced Bcl-2 expression and high IFN-I levels.

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (

The process of antigen recognition depends in part on the amount of peptide antigen available and the affinity of the T cell receptor for a particular peptide-major histocompatibility complex (MHC) molecule complex. The availability of self antigen is limited by antigen processing, which is compartmentalized such that peptide antigens presented by MHC class I molecules originate in the cytoplasm, whereas peptide antigens presented by MHC class II molecules are acquired from the endocytic pathway. This segregation of the antigen-processing pathways may limit the diversity of antigens that influence the development and selection of, e.g., CD4-positive, MHC class II-specific T cells. Selection in this case might involve only a subset of self-encoded proteins, specifically those that are plasma membrane bound or secreted. To study these aspects of immune development, we engineered pigeon cytochrome for expression in transgenic mice in two forms: one in which it was expressed as a type II plasma membrane protein, and a second in which it was targeted to the mitochondria after cytoplasmic synthesis. Experiments with these mice clearly show that tolerance is induced in the thymus, irrespective of antigen compartmentation. Using radiation bone marrow chimeras, we further show that cytoplasmic/mitochondrial antigen gains access to the MHC class II pathway by direct presentation. As a result of studying the anatomy of the thymus, we show that the amount of antigen and the affinity of the TCR affect the location and time point of thymocytes under-going apoptosis.

The requirements for inducing positive selection of T cells were examined in thymus reaggregation cultures, a system in which dispersed populations of immature CD4+8+ cells and purified thymic epithelial cells (TEC) are reaggregated in tissue culture. Studies with TEC from mice selectively lacking major histocompatibility complex (MHC) class I (I-II+), class II (I+II-), or both class I and II (I-II-) molecules showed that class II expression was essential for the differentiation of CD4+8+ cells into CD4+8- cells. Unexpectedly, the generation of TCRhi CD4-8+ cells from CD4+8+ cells was apparent with I-II+ TEC but not with I-II- TEC, perhaps reflecting cross-reactive specificity of CD4-8+ cells for class II molecules. Significantly, the failure of I-II- TEC to generate TCRhi CD4+8- or CD4-8+ cells could not be overcome by adding MHC+ bone marrow-derived cells. These findings, together with experiments on purified subsets of TEC, suggest that positive selection in thymus reaggregation cultures is an exclusive property of cortical TEC.

Cell division during thymic selection was studied with a system in which purified populations of T cell antigen receptor (TCR)- CD4+8+ (double-positive [DP]) cells and fetal thymic epithelial cells (TEC) were reaggregated in tissue culture. In this system, immature DP cells differentiate into mature single-positive (SP) CD4+8- and CD4-8+ TCRhi cells within 3-4 d, indicative of positive selection. By adding the DNA precursor, bromodeoxyuridine, to the cultures and staining cells for bromodeoxyuridine incorporation, T cell division in reaggregation cultures was found to be high on day 1, low on day 2, and high on days 4-5. Cell separation studies established that cell division on day 1 was restricted to DP blast cells. In the absence of blast cells, small DP cells failed to proliferate and differentiated into SP cells without cell division, thus indicating that proliferation is not an essential component of positive selection. This applied to SP cells generated within the first 2-3 d. Surprisingly, the SP cells generated later in culture showed a high rate of cell division; the proliferating SP cells were TCRhi and included both CD4+8- and CD4-8+ cells. Turnover of TCRhi SP cells was also prominent in the normal neonatal thymus and in TEC reaggregation cultures prepared with adult lymph node T cells. We speculate that division of mature SP cells in the perinatal thymic microenvironment is driven by stimulatory cytokines released from TEC. Such proliferation could be a device to expand the mature T cell repertoire before export to the periphery.

Using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to detect cells undergoing early apoptosis, we have defined the surface markers expressed on CD4+CD8+ thymocytes undergoing spontaneous or steroid-induced apoptosis in tissue culture. Some surface markers, e.g., CD4, CD8, and heat stable antigen, are downregulated on apoptotic thymocytes. Surprisingly, however, other markers are upregulated; this applies to T cell receptor beta/CD3, CD69, and CD25 expression. Upregulation of these markers is restricted to a discrete subset of apoptotic cells.

The effector functions of CD4+ cells in vivo are presumed to reflect a combination of lymphokine-mediated bystander reactions and direct cytotoxic T lymphocyte activity. To assess the relative importance of these two mechanisms, we studied the effects of transferring small doses of purified unprimed CD4+ cells to lightly irradiated (600 cGy) recipients expressing major histocompatibility complex class II (Ia) differences. Within the first week after transfer, the host marrow was rapidly repopulated with hemopoietic cells. Thereafter, however, the donor CD4+ cells caused massive destruction of hemopoietic cells, both in marrow and spleen. Marrow aplasia did not affect stromal cells and was prevented by coinjecting donor but not host bone marrow. The use of allotypic markers and fluorescence-activated cell sorter analysis indicated that the destructive effects of CD4+ cells were directed selectively to host Ia+ hemopoietic cells, including stem cells; donor hemopoietic cells and Ia- host T cells were spared. No evidence could be found that the ongoing destruction of host cells impaired the capacity of donor stem cells to repopulate marrow, spleen, or thymus. Moreover, CD4+ cells failed to destroy host-type hemopoietic cells from Ia-deficient mice. Tissue destruction by CD4+ cells thus did not seem to reflect a bystander reaction. We conclude that, under defined conditions, CD4+ cells can manifest extremely potent Ia-restricted CTL activity in vivo, probably through recognition of covert Ia expression on stem cells and/or their immediate progeny.

The previous paper in this series demonstrates that rat T cells developing de novo in the presence of mouse mammary tumor virus (Mtv) antigens in rat-->severe combined immunodeficiency (SCID) mouse xenochimeras display a distinct pattern of V beta-restricted deletion; this deletion pattern is remarkably similar to that occurring during thymic development of mouse T cells in Mtv+ strains. In addition, T cells developing in the absence of Mtv antigens in these rat-->mouse xenochimeras are tolerant of host antigens, but show strong primary proliferative responses in cultures stimulated with Mtv-7+ (Mlsa) mouse cells; like the mouse, these rat T cell responses are dominated by V beta 6 and V beta 8 T cells. Here, we continue analysis of rat T cell responses to superantigens; we show that T cells from Lewis and Fischer 344 rats expressing V beta 8.2 display an important all-or-nothing difference in their responses to Mtv-7 superantigens. This all-or-none strain difference in the response to Mtv-7 applies also to the response by V beta 8.2 and V beta 8.5 T cells to the soluble superantigen staphylococcal enterotoxin B. Because these two rat strains express different alleles of these two V beta 8 family members, this finding identifies additional, hitherto unreported residues of the T cell receptor beta chain important in T cell responses to superantigens.

T cells of mice display V beta-specific reactivity for a spectrum of mouse mammary tumor virus (Mtv) antigens; confrontation with these antigens during ontogeny causes substantial "holes" in the T cell repertoire. Since endogenous Mtv antigens are rare in other species, the question arises whether V beta-specific recognition of Mtv antigens is unique to mice. To examine this question, rat T cells were allowed to differentiate from stem cells in severe combined immunodeficiency (SCID) mice. These rat-->mouse xenochimeras were prepared under a variety of conditions. The results show that rat T cells are strongly reactive to mouse Mtv antigens, both in terms of tolerogenicity and immunogenicity. In fact, the V beta specificity of rat and mouse T cells for Mtv antigens is almost indistinguishable.

Although T cells arise in the thymus, migration of mature postthymic T cells back to the thymus is very limited in adult mice and is restricted to activated cells. In neonates, by contrast, we present evidence that circulating CD4+ and CD8+ T cells with a naive/resting phenotype readily enter the thymus after intravenous injection and remain there for prolonged periods. The migration of resting T cells to the neonatal thymus is largely limited to an unusual subset of cells which lacks expression of the lymph node homing receptor, leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) (MEL-14). Migration of mature T cells to the thymus in neonates may be important for self-tolerance induction.

To test whether unprimed CD8+ cells can recognize class I alloantigens presented selectively on non-bone marrow (BM)-derived cells, unprimed parental strain CD8+ cells were transferred to long-term parent-->F1 BM chimeras prepared with supralethal irradiation. Host class I expression in the chimeras was undetectable on BM-derived cells and, in spleen, was limited to low-level staining of vascular endothelium and moderate staining of follicular dendritic cells (a population of nonhemopoietic cells in germinal centers). Despite this restricted expression of antigen, acute blood-to-lymph recirculation of parental strain T cells through the chimeras led to selective trapping of 95% of CD8+ cells reactive to normal F1 spleen antigen presenting cells (APC) in vitro. Subsequently, a small proportion of the trapped cells entered cell division and gave rise to effector cells expressing strong host-specific CTL activity. The activation of host-specific CD8+ cells was also prominent in double-irradiated chimeras, and cell separation studies showed that the effector cells were generated from resting precursor cells rather than from memory-phenotype cells. It is suggested that the non-BM-derived cells in the chimeras acted as semiprofessional APC. These cells were nonimmunogenic for most host-reactive CD8+ cells but were capable of stimulating a small subset of high-affinity T cells. The possible relevance of the data to the prolonged immunogenicity of vascularized allografts in humans is discussed.

Information was sought on the features of epithelial cells in the murine thymic medulla. The expression of major histocompatibility complex (MHC) molecules on medullary epithelium was defined by light microscopy with the aid of bone marrow chimeras and MHC-transgenic mice. A proportion of medullary epithelial cells was found to show conspicuously high expression of conventional MHC (H-2) class I (K, D, L) and class II (I-A, I-E) molecules. These cells express a high density of the Y-Ae epitope, a complex of an E alpha peptide and I-Ab molecules found on typical bone marrow-derived cells. MHC+ medullary epithelial cells show limited expression of I-O molecules, a class of atypical nonpolymorphic MHC-encoded class II molecules present on B cells. Other medullary epithelial cells express a high density of I-O molecules but show little or no expression of typical MHC class I or II molecules. MHC and I-O expression thus appear to subdivide medullary epithelial cells into two phenotypically distinct subsets. This applies in adults. In the embryonic thymus most medullary epithelial cells express both types of molecules.

Epithelial cells in the thymic medulla are conspicuous in normal adult mice, but sparse in the early fetal thymus and the thymus of adult T cell-deficient SCID mice. To examine whether growth of medullary epithelial cells (MEC) depends upon local contact with mature T cells, we used the finding that the SCID thymus is unusually permeable to mature T cells entering from the bloodstream. When SCID mice received multiple injections of mature lymph node T cells from birth, the thymus accumulated large numbers of mature TCR+ T cells of resting phenotype, but contained virtually no immature (CD4+8+) cells. The injected T cells localized in the medullary region of the thymus and led to marked regeneration of MEC. These and other data suggest that the growth of MEC is under the control of mature T cells. Placing MEC under T cell control might be a device for regulating the size and integrity of the medulla, especially during the phase of rapid thymic growth. Maintaining the cellular components of the medulla in proper balance could be critical for ensuring efficient self tolerance induction.

To seek direct evidence for the notion that stem cells in the thymus need to be constantly replenished from the bone marrow (BM), fetal (day 15) thymuses from normal BALB/c mice were grafted into T and B cell-deficient C.B-17 SCID mice (both H-2d, I-E+). The thymus grafts in these mice showed normal thymopoiesis for the first 3 wk postgrafting but then developed sudden atrophy with near complete loss of CD4+8+ cells by 4-5 wk. Such atrophy was not seen when the thymus-grafted mice were cotransplanted with normal BM cells. The lymph nodes of SCID mice receiving thymus grafts alone contained mature T cells but virtually no B cells. This lack of B cells was associated with aberrant I-E-restricted V beta deletion: the depletion of V beta 3+ and V beta 5+ T cells was near complete, whereas V beta 11+ cells showed only marginal depletion.

To seek information on the potential lifespan of normal B and T lymphocytes, lymph node (LN) cells from unprimed mice were transferred to H-2-identical severe combined immunodeficiency (SCID) hosts. At a population level, the donor B and T cells survived for at least 10 mo post-transfer with no reduction in their numbers. In terms of antibody production, LN-injected SCID mice remained responsive to several different antigens and contained unprimed precursors of memory cells for greater than or equal to 6 mo post-transfer. Most of the B and T cells recovered from LN-injected SCID mice expressed the typical virgin phenotype of mature lymphocytes from young mice. These findings suggest that many of the transferred lymphocytes might have remained in interphase as virgin cells from the time of injection. This did not apply to all of the transferred cells, however, because 20-40% of CD4+ cells from long-term SCID hosts displayed a memory phenotype, 7% incorporated 2-bromodeoxyuridine over 5 d, and total numbers of B and T cells increased gradually (twofold) over a 10-mo period. Collectively, the data favor the view that the pool of mature B and T cells in adult mice is largely self sufficient: some of the cells proliferate, presumably in response to environmental antigens, but many mature cells can remain quiescent for prolonged periods. Input of new cells from the primary lymphoid organs continues, but at a much reduced rate relative to young life.

Long-term H-2-heterozygous a----(a x b)F1 bone marrow (BM) chimeras prepared with supralethal irradiation (1,300 rad) are devoid of Ia+ host BM-derived antigen-presenting cells (APC), but show quite strong host Ia expression in germinal centers, probably on follicular dendritic cells (a class of nonhemopoietic stromal cells). To examine whether Ia expression on these non-BM-derived cells is capable of inducing post-thymic tolerance of T cells, thymectomized irradiated (a x b)F1 mice were reconstituted with parent alpha stem cells and then, 6 mo later, given parent alpha thymus grafts. As measured by primary mixed lymphocyte reactions and V beta expression, the CD4+ cells differentiating in the thymus-grafted mice showed no detectable tolerance to the H-2 (Ia) antigens of the host. To examine whether the thymus-grafted mice contained immunologically significant quantities of host Ia antigens, long-term alpha----(alpha x b)F1 chimeras were injected with normal strain alpha CD4+ cells; the donor cells were recovered from thoracic duct lymph of the chimeras and tested for host reactivity in vitro. The results showed that Ia expression in the chimeras was sufficient to cause selective trapping of a substantial proportion of host-Ia-reactive CD4+ cells soon after transfer and, at later stages, to induce strong priming. Tolerance was not seen. The data place constraints on the view that T cell recognition of antigen expressed on cells other than typical BM-derived APC leads to tolerance induction.

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.