Immunological aspects of aging: early changes in thymic activity

Age related changes in immune function are preceded by changes in thymic function. One index of thymic function is the ability of the thymus to induce the differentiation of bone marrow pre-T cells into active T cells. Movement of precursors of PHA-responsive T cells into the thymus ceases prior to adolescence in long-lived, autoimmune disease resistant mice as determined by studies utilizing chromosome markers in parabiotic mice. The cause for this age-dependent, cell traffic phenomenon is not known, but it would appear that an extrathymic regulatory mechanism may be operating, since these precursor T cells have the capacity to migrate into the thymus, and even a grafted, involuting thymus is capable of accepting precursor T cells, under extraordinary conditions. Results indicate that shortly after adolescence, immature T cell precursors of PHA-responsive cells in the thymus, and perhaps the periphery, are the sole source of PHA-responsive T cells. T cell migration and maturation is discussed in relation to thymic hormones and the thymic-neuroendocrine regulatory axis.     

In rat bone marrow Thy-1 antigen is present on cells with membrane immunoglobulin and on precursors of peripheral B lymphocytes.

Rat bone marrow cells carrying Thy-1 antigen were studied morphologically, and tested for their independence of the thymus and their relationship to the B lymphocyte lineage. Using a fluorescence-activated cell sorter to separate Thy-1⁺ and Thy-1^- fractions, it has been confirmed that up to 50 % of all nucleated bone marrow cells are Thy-1⁺, most of which have the morphology of small lymphocytes. Thy-1^-cells were mainly neutrophils and erythroid. Thy-1⁺ cells were found also in the marrow of B rats (rats thymectomized as adults, irradiated and reconstituted with syngeneic bone marrow from thymectomized donors drained of recirculating lymphocytes), though at a lower frequency (roughly half) than of normal rats. In both normal and B rats about 1/4 of the Thy-1⁺ cells also bore lymphocyte surface immunoglobulin (sIg), and these doubly labeled cells accounted for the majority (～ 2/3) of marrow cells carrying large amounts of sIg. Therefore, unlike mice, Thy-1 is not a marker of thymus-dependent lymphocytes in rats. The B precursor activity of marrow fractions was measured in a long-term re-constitution assay counting sIg⁺ cells in the thoracic duct of lethally irradiated recipients. Virtually all the precursors were in the Thy-1⁺ or sIg^- fractions, and were barely detectable among Thy-1^- or sIg⁺ cells. Thus, in the rat peripheral B lymphocytes descend from precursors bearing Thy-1 antigen but lacking sIg.     

Influence of age on the proliferation and peripheralization of thymic T cells

Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived.     

Restitution of the thymus in lethally irradiated mice after transplantation of syngeneic or allogeneic bone marrow

The early restitution of the thymus of bone marrow chimeras was investigated by the immunoperoxidase technique using monoclonal antibodies against Thy-1 and Lyt-1, Lyt-2, Lyt-3. Within two weeks, normal thymus histology was restored in mice which received untreated syngeneic BM or syngeneic or allogeneic BM pretreated with SAL (specificed antilymphocytic serum). Irradiation depleted the thymic cortex of small Thy-1+, Lyt-1+2+3+ cells but did not affect a medullary population of medium sized weakly stained Thy-1+, strongly stained Lyt-1+ cells. Preceded by the appearance of an increasing number of large Thy-1+, Lyt-1- blasts (days 2 and 4), the thymic cortex was repopulated (beginning on day 6) by smaller Thy-1+ cells which acquired Lyt-1, Lyt-2 and Lyt-3 though, obviously not in a strictly sequential manner. Simultaneously, the medullary radioresistant cells disappeared, nd the medulla was subsequently repopulated (beginning on day 8) by thymocytes of a mature phenotype. Early restitution of the thymus in radiation control mice was similar to the bone marrow chimeras. The results indicate that the histological restitution of the thymus originates substantially from radioresistant precursors of host origin. Graft-versus-host reaction induced by untreated allogeneic bone marrow cells prevented normal thymic restitution. A delayed localized cortical repopulation with small Thy-1+, Lyt-1+2+3+ cells, progressive destruction of thymic architecture and almost no restoration of the medullary immunocompetent thymocytes were noted. T cell differentiation obviously was seriously affected by the injuries to the thymic microenvironment due to alloreactive T cells.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with (1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; (2) Mococlonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and (3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: (1) the majority of cortical thymocytes are strongly Thy-1⁺ (positive), strongly T-200⁺, variable in Lyt-1 expression, and strongly Lyt-2⁺; (2) the majority of medullary thymocytes are weakly Thy-1⁺, strongly T-200⁺, strongly Lyt-1⁺, and Lyt-2^? (negative); (3) a minority of medullary cells are weakly Thy-1⁺, T-200⁺, strongly Lyt-1⁺, and strongly Lyt-2⁺; (4) a small subpopulation of subcapsular lymphoblasts is Thy-1⁺, T-200⁺, and negative for the expression of Lyt-1 and Lyt-2 antigens; (5) a small subpopulation of subcapsular lymphoblasts is only Thy-1⁺ but T-200^? and Lyt^?; and (6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Graft-Versus-Host Reactions in Mice: III. Epithelioid and Multinucleated Giant Cells of Thymic Origin

Thymus cells from C3H donor mice were infused into lethally irradiated (C3H × C57BL/10)F₁ hybrid mice. Light and electron microscopic examination of the lymphoid tissues of recipients revealed the presence of epithelioid and multinucleated giant cells in addition to lymphocytes and histiocytes. Serial transplantation of thymus-derived cells into irradiated F₁ hybrid mice resulted in a preponderance of epithelioid cells over lymphocytes. Epithelioid cells, as well as lymphocytes and histiocytes, incorporated ³H-thymidine, indicating that they were actively proliferating. Following transplantation of CBA-T6T6 thymocytes into irradiated (C3H × C57BL/10)F₁ mice, karyotypic analysis of host lymphoid tissues indicated that all dividing cells were of donor origin. Whereas bone marrow is known to give rise to epithelioid cells in the absence of thymic influences, the thymus must also contain precursors of epithelioid cells.     

Jagged-1hi or Delta-1hi thymic stromal cell populations can trigger acquisition of Thy-1 and loss of B220

Effects of endogenous Notch ligands Jagged-1 and Delta-1 on maturation markers in adult mouse lymphocytes were checked in vitro on putative thymocyte precursors from bone marrow and on DN3 cells from the thymus. Both populations were cocultured with stromal cells (Small M, Histochem Cell Biol 2005;123:513) naturally expressing high levels of either Jagged-1 or Delta-1. Initially the bone marrow cells were Thy-1.2(neg) while the majority were B220(hi). These trends were reversed by culture on both the Jagged-1(hi) and Delta-1(hi) monolayers. Then DN3 thymocytes were cultured with stroma expressing these two ligands and the T cell receptor beta chain was expressed more clearly after coculture on Delta-1(hi) stroma. These findings indicate that stromal cells with endogenous Jagged-1 as well as Delta-1 could initiate early changes toward T cell commitment while Delta-1 favored later alphabeta-TCR development.     

The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens.

Rat lymphoid cells have been labeled with sodium 3H-borohydride after periodate oxidation. The labeled glycoproteins were solubilized in detergent and analyzed by fluorography after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Major bands were found at 150 000, 95 000 and 25 000 apparent mol.wt. for thymocytes; at 170 000 and 95 000 mol. wt. for T lymphocytes and at 200 000 mol.wt. for B lymphocytes. Bone marrow cells showed a diffuse band at 100 000 mol.wt. with relatively minor bands around 150 000 mol.wt. With the exception of the 95 000 mol. wt. bands, all these glycoproteins bound to lentil lectin. Using monoclonal or monospecific antibodies in immunoprecipitation and on antibody affinity columns, each of these glycoprotein bands was identified as a previously defined lymphocyte differentiation antigen. The bands at 150 000 mol.wt. on thymocytes, at 170 000 on T lymphocytes, at 200 000 on B lymphocytes, and at 130 000 to 150 000 on bone marrow cells all consist of a leukocyte-common antigen, which has previously been shown to be present on leukocytes but not on other tissues. At least a part of the 95 000 mol.wt. band on thymocytes, T lymphocytes and bone marrow cells is the W3/13 antigen previously shown to be on mature T lymphocytes, polymorphonuclear cells, and in brain. The 25 000 mol.wt. band of thymocytes is the Thy-1 antigen. Similar experiments were carried out on thymocytes labeled with ¹²⁵I by the lactoperoxidase method. An intense band at 150 000 mol. wt. was identified as the leukocyte-common antigen by immunoprecipitation. A labeled band, which did not bind to lentil lectin, was immunoprecipitated at 95 000 mol. wt. with W3/13 antibody. Rat Thy-1 antigen was not labeled with ¹²⁵I.     

Postirradiation thymocyte regeneration after bone marrow transplantation. III Intrathymic differentiation and development of thymocyte subpopulations

Fluorescence distributions of thymocytes stained for Thy-1 as well as size measurements were used to discriminate between thymocyte subpopulations during regeneration of the thymus after irradiation and bone marrow transplantation. Subpopulations with “low” and “high” Thy-1 density of donor- and recipient-derived progeny were quantitated. They were continuously present in the thymus and developed simultaneously but at different rates of growth. A similar developmental pattern was observed for donor- and host-derived “high” Thy-1⁺ cells, whereas “low” Thy-1⁺ cells of donor and recipient origin showed markedly different growth patterns. This indicated that development of the two subpopulations took place independently. During early regeneration donor-derived “low” and “high” Thy-1⁺ cells contain a high proportion of large cells, indicating the presence of cycling cells in both subpopulations.     

The combined loss of Gads and CD127 reveals a novel function of Gads prior to TCRβ expression

The Gads adaptor protein is an essential component of the T cell signaling complex critical for T cell receptor-mediated calcium mobilization. After expression of TCRβ in T cell precursors, Gads is required for optimal Bcl-2 expression and cell survival. Similarly, the IL-7 receptor chain CD127 is also necessary for optimal Bcl-2 expression and cell survival in TCRβ-expressing thymocytes. Based on these observations, we tested whether Gads and CD127 might regulate convergent or linear signaling pathways by crossing Gads^?/? mice with CD127^?/? mice. Thymi from Gads^?/?CD127^?/? mice were barely detectable and many of the thymocytes were within the DN1 population. By contrast, B cell development in the Gads^?/?CD127^?/? mice was comparable to that of CD127^?/? mice, indicating that the combined loss of Gads and CD127 did not lead to a global deficit in hematopoiesis. Analysis of Lin^?Sca-1⁺c-kit⁺ bone marrow cells and bone marrow chimera experiments indicated that Gads^?/?CD127^?/? T cell precursors either failed to migrate into the thymus or survive in the thymus. These data demonstrate that Gads functions at a stage of T cell development that had not been previously described.     

Expression of T cell receptor V gamma 5 in the adult thymus of irradiated mice after transplantation with fetal liver cells

T cell receptor (TcR) gamma/delta displays limited diversity and its diversity is distinct in different stages of ontogeny and in different anatomical sites. The V gamma 5 and V delta 1 gene products are preferentially expressed on the early fetal thymocytes and on Thy-1+ dendritic epidermal cells, whereas the V gamma 4 and V delta 5 gene products are abundantly expressed on the adult thymocytes. To elucidate whether the developmentally ordered appearance of thymocytes expressing TcR gamma/delta is dependent on the source of T cell precursors or is controlled by the thymic environment where T cells develop, we compared the expression of V gamma 5 on the early-appearing thymocytes between irradiated mice after transplantation with fetal liver (FL) cells and those after transplantation with bone marrow (BM) cells. Sequential appearance of thymocyte subpopulations was observed in the thymus of radiation FL chimeras similar to that seen in radiation BM chimeras. A substantial number of thymocytes bearing V gamma 5 appeared in the thymus at the early stage of radiation FL chimeras, whereas few, if any, of such V gamma 5-bearing thymocytes were detected in the thymus at any stage of radiation BM chimeras. These results suggested that the ordered expression of V gamma repertoire may depend on the origin of the T cell precursors but not on the thymic environment.     

Thymic nurse cells and thymic repopulation after whole body sublethal irradiation in mice

Thymic Nurse Cells (TNCs) are lymphoepithelial complexes which are thought to play a role in the early stages of the intrathymic differentiation pathway. Therefore, their repopulation kinetics were analyzed in mice after sublethal whole-body irradiation. Changes of the number of TNCs per thymus were parallel with the evolution of the whole thymocyte population. Particularly, a first wave of TNCs restoration was followed by a secondary depletion and a final recovery. This suggests that TNCs restoration is related to the proliferating progeny of intrathymic radioresistant thymocytes. When normal bone marrow cells were grafted intravenously after irradiation, no secondary depletion was found. This pattern of restoration was obviously related to thymic repopulation by cells which were derived from the inoculated bone marrow. Homing studies with FITC labelled bone marrow cells showed that inoculated bone marrow cells did not penetrate TNCs early after irradiation. Later on, when immigrant cells started to proliferate, they were found preferentially within TNCs before spreading in the whole thymus. The results indicate that interactions between immature thymocytes and epithelial cells within TNCs are critical for the first steps of intrathymic lymphopoiesis.     

Cell interactions in the induction of tolerance: the role of thymic lymphocytes

Thymectomized, lethally irradiated, bone marrow reconstituted mice were treated with a large dose of sheep red blood cells (SRBC) over the course of 30 days. They were unable to respond to further antigenic challenge for one month. Fifteen million thymocytes given 4 days after the termination of treatment restored their ability to respond.

The same antigenic treatment given to similar chimeras, which differed only in having had 15 × 10⁶ thymus cells added to the bone marrow inoculum, also abolished the response to further antigenic challenge. In contrast to chimeras without thymus cells present during the course of treatment, the later addition of thymocytes to these animals did not restore their response. It did, however, restore the response to a second challenge of antigen given 17 days after the addition of thymocytes. This response was the same as non-treated animals given only one injection of thymocytes and significantly less than non-treated animals given thymocytes twice.

The following explanation of these results is offered. Bone marrow derived (BMD) lymphocytes that can make antibody without assistance of thymus derived (TD) lymphocytes were made tolerant in the absence of TD cells. Thymus dependent BMD cells were not. New cells, coming from the bone marrow, broke the tolerant state within a month.

When TD cells were present both populations of BMD cells, as well as the TD cells, were made tolerant. New BMD cells regenerating from the bone marrow abrogated the tolerant state of the BMD population. This breaking of tolerance could only be seen in mice given additional thymocytes as the tolerance of the TD cells was not broken in the absence of a thymus.

Thus, the induction of tolerance as well as the induction of immunity in thymus dependent BMD cell populations, seems to require the co-operation of TD cells.

     

Many cells in rat bone marrow have cell-surface Thy-1 antigen.

Thy-1.1 and Thy-1 xenoantigenic determinants were detected at the cell surface of many rat bone marrow cells. The absorptive capacity of bone marrow cells was 6-10% of that of thymocytes for Thy-1 antigenic determinants, and 30-45% of rat bone marrow cells were specifically labeled with anti-Thy-1 antibody as detected by autoradiography. Thus, while mice and rats are similar in having large amounts of Thy-1 in brain and thymocytes, they differ in that the rat lacks the antigen in most peripheral T cells and expresses it in a large number of bone marrow cells; the opposite is true in the mouse.     

Low level of mixing of partner cells seen in extrathymic T cells in the liver and intestine of parabiotic mice: its biological implication

c-kit⁺ stem cells have recently been found in the liver and intestine of adult mice. We examined whether such stem cells give rise to extrathymic T cells in these organs in situ. To this end, we used parabiotic B6.Ly5.1 and B6.Ly5.2 mice, i.e. mice sharing the circulation. The origin of lymphocytes was identified by anti-Ly5.1 and anti-Ly5.2 monoclonal antibodies in conjunction with immunofluorescence assays. Lymphocytes in the blood, spleen, lymphnodes and liver had become a half-and-half mixture of Ly5.1⁺ and Ly5.2⁺ cells in both individuals by day 14. However, this level of mixing decreased in extrathymic T cells in the liver ( i.e. NK T cells) and intestine by day 14 and thereafter. The same was observed in T cells of the thymus. The data from immunohistochemical staining supported the results of immunofluorescence assays for suspension cells. The present results raise the possibility that extrathymic T cells in the liver and intestine may arise from their own pre-existing precursor cells, possibly from their own stem cells. Another important finding was that the composition pattern of lymphocyte subsets in one individual was quite similar to that in its partner at various sites. This result was interpreted to mean that only selected partner cells migrate to specific sites in the other partner individual.     

Effect of hydrocortisone on immune system of the lizard,Chalcidesocellatus II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, $\text{Chalcides}$$\text{ocellatus}$ were cultured for 24 hr in the presence of 10^?3M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30–50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult $\text{Ch}$. $\text{ocellatus}$ lead to depletion of all TSA⁺ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.     

Patterns of dual lymphocyte development in co-cultures of foetal thymus and lymphohaemopoietic cells from young and old mice.

Patterns of lymphocyte development in the thymus were analysed, focusing on newly emigrating bone marrow (BM) and resident thymic cells. We co-cultured foetal (Day 15 of gestation) thymic explants (FT, C57BL/Ka, Thy-1.1), with BM cells from young (2-3 months) or old (24 months) syngeneic, Thy-1 congenic (C57BL/6J, Thy-1.2) mice. When the FT was severely depleted [treated with either 2-deoxyguanosine (dGua) or exposed to an irradiation dose of 20 Gy] BM-type T lymphocytes were dominant, regardless of BM donor age. When the FT was only partially depleted of its proper lymphoid cells (by exposure to 10 Gy), the lymphocytes which developed were from both BM and FT origins, yet the level of donor-type thymocytes from the young mice was higher than that of the old. Under these conditions the proportion of FT-derived double-positive CD4+ CD8+ (DP) cells was higher, and that of single-positive CD4- CD8+ cells was lower, than in the BM-derived cells, irrespective of the BM donor age. The proportions of old BM-derived DP cells were lower than in the young. Co-cultures of thymus cells from young and old mice with partially depleted FT explants resulted in similar proportions of CD4/CD8 subsets from both donor and FT origins, with the exception that in the presence of old-thymus cells there was an increase in the level of FT-type CD4- CD8+ cells. Patterns of T-cell differentiation in the thymus thus seem to be determined by newly emigrating cells and the resident thymocytes.     

Effect of hydrocortisone on immune system of the lizard, II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, were cultured for 24 hr in the presence of 10⁻³M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30–50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult . lead to depletion of all TSA⁺ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.     

Flow microfluorometric analysis of mouse thymus development in vivo and in vitro

The phenotypic properties of lymphoid cells in the developing embryonic thymus were characterized using monoclonal antibodies and flow microfluorometry. CBA/J-T6/T6 thymocytes stained with antibodies directed against Thy-1.2, Lyt-1, Lyt-2 or H-2K^k were simultaneously analyzed for fluorescence intensity and forward light scatter (FLS), a cell size-related parameter. Whereas Thy-1 and Lyt-1 antigens were already present on 15-day fetal thymocytes, Lyt-2 expression was first detectable on day 16 and increased rapidly thereafter to reach adult levels by day 19. Concomitant with these phenotypic changes, rapid changes in FLS occurred during this time period. The FLS distribution of Lyt-2⁺ cells was initially homogeneously high (day 16) but became biphasic at days 17–18. Thereafter, the lower FLS subpopulation predominated. FLS changes in Lyt-2^? cells could be dissociated kinetically from changes in the Lyt-2⁺ subpopulation. Thus high FLS Lyt-2^? cells were the predominant subpopulation throughout the entire fetal period and could still be detected after birth, when a population with lower FLS first appeared. The embryonic thymus developing in vivo was then compared with the 13-day embryonic thymus maintained for 14 days in an in vitro organ culture system. Based on a combination of fluorescence and FLS analysis, the organ-cultured thymus appeared to share certain phenotypic properties with the 18–19 day in vivo developing thymus.     

Thymopoietic and bone marrow response to murine Pneumocystis pneumonia

CD4⁺ T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4⁺ T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9⁺ multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4⁺ T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4⁺ cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4⁺ T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4⁺ T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9⁺ MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9⁺ MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4⁺ T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.