Age-dependent loss of naïve T cells in TCR transgenic bone marrow chimeras

Study of immune senescence is complicated by low numbers of antigen-specific lymphocytes, particularly na?ve T (Tn)cells which disappear with aging. Although T cell receptor (TCR) transgenic mice facilitate aging research, their large number of Ag-specific T cells renders their T cell pool abnormal, precluding normal in vivo immunity. To create a physiologically relevant model with measurable numbers of TCR transgenic CD4+ T cells in the context of normal lymphocytes, mixed (DO11.10+BALB/c) bone marrow (BM) chimeras were constructed. As found in normal mice, the total number of transgenic T cells and the Tn:memory T cell ratio declined with the aging of the BM chimeras. Although these shifts in T cell subsets were evident in both the lymph nodes and the spleen (SP), they were more pronounced in the SP. Unlike DO11.10 mice, transgenic T cells in the chimera acquired an effector/memory phenotype upon antigen challenge. These results reveal a pliable model to study the impact of the constriction of the Tn cell repertoire upon optimal vaccine responses in the elderly.     

Cytotoxic reactivity of gut lamina propria CD4+ αβ T cells in SCID mice with colitis

Polyclonal, mucosa-seeking memory/effector CD4⁺ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe-combined immunodeficient (SCID) mice developing colitis after CD4⁺ T cell transplantation. CD4⁺ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)-2 + IL-7. CD3 ligation enhances this cytokine-supported proliferation in CD4⁺ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine-supported proliferation in CD4⁺ T cells from the gut lamina propria in a cell density- and dose-dependent manner. Almost all CD4⁺ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4⁺ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4⁺ T cells show Fas-dependent cytotoxicity. A large fraction of gut lamina propria CD4⁺ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4⁺ T cells are apoptotic. Hence, a large fraction of colitis-inducing CD4⁺ T cells undergo activation-induced cell death in situ and can damage other cells through Fas-dependent cytotoxicity.     

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Changes in the Intestinal Lymphoid Compartment Throughout Life: Implications for the Local Generation of Intestinal T Cells

The intestinal lymphoid compartment has a rather stable composition throughout life. However, both during early neonatal development and at high age unique cell populations can be recognized at distinct sites in the intestinal tissue. Directly after birth all intestinal CD3⁺ cells are found in the lamina propria. At this time the epithelium does not contain T cells. These CD3⁺ lamina propria lymphocytes co-express both TCRβ and TCRδ chains, probably reflecting the expression of a TCRβδ heterodimer on the cell surface. Cells with this particular phenotype are present in comparable numbers in the lamina propria of both neonatal euthymic and athymic mice, indicating the thymus-independent nature of these cells. During aging the frequency of TCRαβ⁺ CD8αα⁺ intestinal T cells increases. These cells are also considered to be thymus-independent. The appearance of high numbers of CD4⁺ CD8αα⁺ intestinal T cells in aged mice is even more striking. It is postulated that the neonatal TCRβδ⁺ cells, and probably also the CD4⁺ CD8αα⁺ cells as found in old mice, are intermediates in the extrathymic differentiation pathway of TCRαβ⁺ CD8αα⁺ intestinal T cells.     

Effects of the lpr/lpr mutation on T and B cell populations in the lamina propria of the small intestine, a mucosal effector site.

MRL mice, which develop a lymphoproliferative disease characterized by increased numbers of alpha/beta T cell receptor+ (TCR+) B220/6B2+CD4-CD8- T cells [lymphoproliferation (lpr) T cells], were studied for the effect of the lpr/lpr mutation on the mucosal immune system in the gastrointestinal (GI) tract. We analyzed the effect of the lpr gene mutation on T and B cell populations in the Peyer's patches (PP) and the lamina propria lymphocytes (LPLs), as examples of major IgA inductive and effector tissues in the GI tract respectively. Normal mouse PP contain B cells committed to IgA (surface IgA+) but only low numbers of B cells producing IgA. However, enhanced spontaneous IgA and IgG synthesis occurs in the PP of MRL mice. Further, we have now shown that PP of MRL mice are populated by lpr T cells. Interestingly, lpr T cells were not present in significant numbers in LPLs of MRL mice, even in older animals. Of interest was the finding that the ratio of CD4+ to CD8+ T cells in the lamina propria was lower in MRL when compared with control mice, and the CD8+ T cell subset actually predominates in LPLs of autoimmune mice. In addition, the number of gamma/delta TCR+ T cells in LPL of MRL lpr/lpr mice was significantly increased, especially in MRL lpr/lpr mice at 6 and 12 weeks of age. When the isotype distribution of B cells in LPLs was analyzed, no changes were noted in MRL lpr/lpr mice in comparison with MRL +/+ or normal control mice, and the pattern was IgA much greater than IgM greater than IgG. These results show that although increased numbers of CD8+ T cells and gamma/delta TCR+ cells occur in the LPLs of MRL mice, a normal distribution of plasma cell isotypes (IgA much greater than IgM greater than IgG) is found in this mucosal compartment. Further, Ipr T cells do not develop in the lamina propria compartment of the GI tract.     

Intestinal helminth infection induces highly functional resident memory CD4+ T cells in mice

Immunity to intestinal nematodes requires CD4? Th2‐cell responses, including IL‐4 and IL‐13 production. Chronic infection with intestinal nematodes leads to downregulation of these responses, and few functional T helper (Th) 2 cells are detected in secondary lymphoid organs in the chronic phase or after abrogation of infection. Here, we show with a natural murine infection with Heligmosomoides polygyrus that highly functional memory Th2 cells persist in the lamina propria and in addition in the peritoneal cavity (PC) after abrogation of infection. While both tissue‐resident memory (T_RM) populations proliferate in situ and express IL‐4, IL‐5, and IL‐13 upon TCR‐dependent stimulation, only peritoneal memory cells express high levels of the IL‐33 receptor and produce IL‐5 and IL‐13 upon TCR‐independent stimulation with IL‐33 and IL‐7. Most importantly, PC‐derived T_RM cells are able to mediate anti‐helminthic effects by decreasing the fecundity of female worms upon transfer into recipient mice. These results show that nonlymphoid compartments can serve as reservoirs for Th2 memory cells, and furthermore that innate effector function of Th2 memory cells is restricted to CD4? memory T cells residing in the PC.     

Intestinal intraepithelial lymphocytes preferentially repopulate the intestinal epithelium

We have used C.B-17 severe combined immune deficiency (SCID)mice to study the repopulation of intestinal intraepithelialcells in these mice. We have found that intestinal intraepitheliallymphocytes (IELs) injected into SCID mice preferentially repopulatethe intestinal epithelium. About 5 weeks after injection wecan detect significant numbers of IEL in repopulated SCID mice.Repopulation occurs in {small tilde}70% of the injected miceand the amount of recovered cells per mouse is variable. Therecovered cells are of donor-type origin and exhibit a typicalIEL phenotype. The donor-type T lymphocytes that can sometimesbe found in other organs of IEL-repopulated SCID mice are generallyof low number. They are not stained with antibodies againstIEL-specific markers and their phenotypes appear to be moretypical for T cells normally found in these sites. In contrast,the intestinal epithelium of SCID mice cannot be efficientlyrepopulated with lymphocytes using cells of other organs includingthymocytes, Peyer's patch lymphocytes, and bone marrow cells.From our data we conclude that intestinal IELs are confinedto the intestinal epithelium and possibly contain a precursor-typecell that preferentially regenerates cells of its own population.     

Immunosenescent colitogenic CD4(+) T cells convert to regulatory cells and suppress colitis

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4(+) cells. However, it remains unknown whether colitogenic CD4(+) cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4(+) cells, we performed sequential transfers of LP CD4(+) cells from colitic CD4(+)CD45RB(high) cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4(+) cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4(+) cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-gamma, TNF-alpha, and IL-17 than colitic LP CD4(+) cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4(+) cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4(+)CD45RB(high) cells. Thus, colitogenic LP CD4(+) cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence.     

Regulatory T cells require renal antigen recognition through the TCR to protect against injury in nephritis

Regulatory T cells (Treg) are important for maintaining immune homeostasis. Adoptive transfer of Tregs is protective in renal disease models in both immunocompetent and immunodeficient mice. However the involvement of TCR recognition of renal antigens remains to be clarified. To address this question, we made use of Tregs from the DO11.10 mouse (a TCR transgenic (Tg) mouse), that recognise the non-murine antigen Ovalbumin (OVA) and therefore are not activated by renal antigens. DO11.10 Tregs were assessed functionally in vitro and demonstrated equivalent suppression to WT BALB/c Tregs. Adriamycin Nephropathy (AN) was induced in mice which had been transfused with CD4⁺CD25⁺Tregs isolated from DO11.10 or BALB/c mice. To eliminate the memory/activation state as a cause of differences in activity, the protective capacity of DO11.10 Tregs pre-activated with OVA in vivo was assessed. Transfer of WT BALB/c Tregs significantly attenuated the development of AN with less glomerulosclerosis, tubular atrophy and macrophage infiltration as compared to AN mice without Treg transfer. However, mice receiving either naïve or pre-activated DO11.10 Tregs were not protected from AN. The lack of protection by DO11.10 Tregs was not due to failure to traffic to the affected kidney. These results suggest that antigen recognition in the kidney is important for Treg protection against injury.     

Development of T cells expressing an altered TCR complex

Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.     

CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance

CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required.     

Bcl6 is essential for the generation of long-term memory CD4+ T cells

Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells. We analyzed here a role for Bcl6 in the generation of long-term memory CD4(+) T cells. Naive CD45RB(+) CD4(+) T cells from Bcl6-deficient DO11.10 (KJ1.26(+)) transgenic mice were transferred into BALB/c mice and immunized with ovalbumin peptide and LPS. Long-term memory KJ1.26(+) CD4(+) T cells from wild-type mice were detected in the spleen, lungs and liver during 10 weeks after immunization; however, Bcl6-deficient KJ1.26(+) CD4(+) T cells were vanished completely in those organs 4 weeks after immunization. Since memory CD4(+) T cells can be generated from effector CD4(+) T cells, properties of Bcl6-deficient effector CD4(+) T cells were compared with those wild-type effector CD4(+) T cells 10 days after immunization. Numbers of IFN-gamma-non-producing CD45RB(-), CD62L(+) or IL-7Ralpha(+) effector CD4(+) T cells in the spleen, lungs and liver were similar between Bcl6-deficient and wild-type CD4(+) T cells. However, the percentage of apoptotic cells in Bcl6-deficient effector CD4(+) T cells was higher than that in wild-type effector CD4(+) T cells. At the late effector phase, the number of IFN-gamma-non-producing cells and the percentage of apoptotic cells in Bcl6-deficient CD4(+) T cells were smaller and higher than those in wild-type CD4(+) T cells, respectively. These data suggest that Bcl6 in CD4(+) T cells plays a role in protection of memory precursor CD4(+) T cells from apoptosis and may involve in survivability of long-term memory CD4(+) T cells.     

Lamina Propria T Cell Subsets in the Small and Large Intestine of Euthymic and Athymic Mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C. B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/ xid) mice. CD3⁺ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20–30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the αβ lineage in euthymic mice, but only 40% were of the αβ lineage in athymic mice. T cells of the αβ lineage were thus more frequent than T cells of the αβ lineage in the intestinal lamina propria T cells of extrathymic origin. CD4⁺ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20–30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8⁺, In intestinal lamina propria T cell populations of athymic mice, the CD8⁺ T cell population was expanded. Most (60–70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8α⁺β^? form of the CD8 coreceptor. A fraction of 15–20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were ‘double negative’ CD4^? CD8^?. A large fraction of the TCRαβ⁺ T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

CD4+CD8+ murine intestinal intraepithelial lymphocytes

We have studied a population of CD4+ intestinal intraepithelial lymphocytes using two-color flow cytometric analyses, and in highly purified fluorescent-activated cell-sorted preparations. Although CD4+ T cells present within the epithelial immune compartment comprised only approximately 10-20% of the total intestinal epithelial lymphoid cells, an unusually high proportion of those CD4+ lymphocytes expressed a CD4+CD8+ phenotype which is rarely encountered in peripheral T cells. By comparison, CD4+ lymphocytes from spleen or lymph nodes existed exclusively as single-positive T cells. Analyses of CD4 and CD8 expression on lymphocytes from Peyer's patches, the lamina propria, and IEL indicated that CD4+CD8+ lymphocytes were unique to the IEL. Using CD4+CD8+ preparations obtained by fluorescent-activated cell sorting, CD4+CD8+ epithelial T cells were found also to express CD3 and Thy-1 surface markers. This heretofore undescribed extrathymic population of double-positive T cells constitutes a unique peripheral T cell subset which may be involved in intestinal T cell maturation and development, or could represent a highly specialized effector population.     

Phenotype and function of lamina propria T lymphocytes

Lamina propria T cells have a low expression of the CD45RA antigen and a high expression of the CD45RO antigen. This phenotype is characteristic for memory T cells (table 2). In addition, T cells in the effector compartment of the mucosa bear surface antigens which are very rarely found in other sites of the immune system. Intestinal T cells also express functional IL-2 receptors and IL-2 receptor alpha chain mRNA, and are able to synthesize high amounts of IL-2. However, another marker of memory T cells, CD29, is not expressed in high density in the lamina propria indicating that lamina propria T cells differ from 'classical' memory T cells. This is supported by functional studies in nonhuman primates infected rectally with C. trachomatis which show that lamina propria T cells do not proliferate after stimulation with antigen but rather provide helper function for immunoglobulin synthesis (table 2). The intestinal lamina propria therefore contains highly specialized T cells which have a distinctive phenotype and are activated. Functionally these T cells can be characterized as differentiated effector lymphocytes which respond to triggering the antigen-specific T cell receptor by secreting helper factors for B cells. This concept is supported by recent studies showing that the pattern of lymphokines produced by lamina propria T cells and the responsiveness to certain lymphokines differ from those of other lymphocyte populations [25]. Lamina propria T cells thus represent a subset of memory T cells with a unique maturational state.     

Restriction of the CD4+ T-cell receptor repertoire prevents immune pathology in TGF-beta1 knockout mice

Mice with a targeted deletion in TGF-beta1 spontaneously develop CD4+ T-cell-dependent multifocal inflammatory disease and autoimmune pathology. T cells from TGF-beta1-/- mice are strongly activated, but the mechanisms that lead to T-cell activation and organ pathology are not well understood. Recent work shows that TGF-beta1 raises the threshold for signaling through the TCR, suppressing the response of T cells to mitogenic stimuli. This suggests the possibility that CD4+ T cells in TGF-beta1-/- mice become aberrantly activated and cause damage in response to physiologic inputs that ordinarily are not sufficient for cell activation, such as homeostatic MHC-TCR interactions, cytokines, or adhesion molecules. This model predicts that pathology is largely antigen-independent, and that CD4+ T cells, regardless of antigen specificity, will become activated in TGF-beta1-/- mice, with subsequent organ pathology. To test this model, we crossed BALB/c-TGF-beta1-/- mice with the DO11.10 TCR transgenic mouse. To obviate the possible development of nonclonotypic TCRs, we also bred in a deficiency in RAG-1. Cohorts of highly inbred BALB/c background TGF-beta1-/- mice with an increasingly restricted CD4+ T-cell repertoire (TGF-beta1-/- mice; DO11.10-TGF-beta1-/- mice; DO11.10-RAG-1-/-TGF-beta1-/- mice) were then analyzed for inflammatory organ pathology and T-cell activation. The data show that progressively restricting the CD4+ T-cell repertoire improved survival, ameliorated target organ pathology, and reduced T-cell activation to control levels. Therefore, these results find no support for the involvement of atypical T-cell activation pathways in disease in TGF-beta1-/- mice. Rather, T-cell activation and pathology in TGF-beta1-/- mice appear to be functions of typical TCR activation pathways. This supports the hypothesis that immune pathology in TGF-beta1-/- mice is self-antigen triggered.     

"Bystander polarization" of CD4+ T cells: activation with high-dose IL-2 renders naive T cells responsive to IL-12 and/or IL-18 in the absence of TCR ligation

Responsiveness of CD4+ T cells to the IFN-gamma-inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4+ T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4+CD62L+ Tcells from normal C57BL/6 mice or from DO11.10/Rag2(-/- )OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-gamma when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4+ T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4+ T cells from DO11.10/Rag2(-/- )mice exhibited polarization towards a Th1 phenotype (high IFN-gamma but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term "bystander polarization" to describe this phenomenon and we speculate that bystander polarization of naive CD4+ T cells may occur in vivo during strong antigen-specific immune responses.     

T lymphocytes in host defense against bacterial translocation from the gastrointestinal tract.

Flow cytofluorometric analyses of lymphocytes harvested from the mesenteric lymph node (MLN), mucosal epithelium, and lamina propria of C57BL/6 mice demonstrate that expression of alpha/beta or gamma/delta T-cell receptors (TCR) and CD4 or CD8 molecules by T lymphocytes in the intestinal immune system varies depending upon their anatomic location. The MLN contained equivalent numbers of CD4+ and CD8+ T cells, the vast majority of which were alpha/beta TCR positive (alpha/beta TCR+). The lamina propria T cells were predominantly CD4+ and alpha/beta TCR+, while the intestinal intraepithelial lymphocytes consisted of equivalent numbers of alpha/beta and gamma/delta T cells, the majority of which were CD8+. There were no significant changes in these T-cell phenotypic profiles when the mice were antibiotic decontaminated or monoassociated with Escherichia coli. Mice were depleted of CD4+ T cells and/or CD8+ T cells in vivo by intraperitoneal injections of monoclonal antibody GK 1.5 (rat anti-mouse CD4) and/or monoclonal antibody 2.43 (rat anti-mouse CD8). T-cell depletion was confirmed in the MLN, lamina propria, and the intestinal epithelium by flow cytometry. E. coli C25 translocation from the gastrointestinal (GI) tract to the MLN was significantly increased in mice depleted of CD4+ T cells, CD8+ T cells, or both. T-cell-deficient athymic beige/nude mice also exhibited greater levels of E. coli C25 translocation to the MLN than beige/het euthymic littermates. Salmonella typhimurium translocation also was increased following CD4+ and CD8+ T-cell depletion in mice monoassociated with S. typhimurium. Depletion of CD4+ and/or CD8+ T cells also increased the translocation to the MLN of certain indigenous GI flora bacteria. These results confirm that T-cell-mediated immunity is involved in the host defense against bacterial translocation from the GI tract.     

Localization of IFN-gamma and IL-6 mRNA in murine intestine by in situ hybridization.

Previous studies have highlighted the importance of CD4+ T cells in regulation of IgA responses and have indicated a functional heterogeneity among these cells between inductive (Peyer's patch) and effector (lamina propria) sites in the intestine. To determine whether these functional differences could be accounted for by differences in cytokine profile of cells in each of these sites, the distribution of mRNA for interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) was investigated by in situ hybridization techniques using 35S-labelled riboprobes. Whereas message for IL-6 is abundant in all regions of the lamina propria from the base of the mucosa to the tips of the villi, very little is expressed in Peyer's patches or in the epithelium. In contrast, message for IFN-gamma is expressed predominantly by cells localized only in the base of the lamina propria and, as with IL-6, very little message was detected in Peyer's patches although occasional strongly positive IFN-gamma cells were observed in the epithelium. These results indicate that, at least in the absence of deliberate intestinal stimulation, functional expression of these cytokines is restricted to effector rather than induction sites in the intestine. This is consistent with our previous observations demonstrating a requirement for T-cell signals in promoting post-extravasation differentiation and proliferation of IgA-committed B cells in vivo and the implications of these findings to the role of the Th1 and Th2 subsets of CD4+ cells in mucosal immune responses is discussed.