CD43 potentiates CD3-induced proliferation of murine intestinal intraepithelial lymphocytes

The involvement of CD43 in cell proliferation of murine intestinal intraepithelial lymphocytes (IEL) has been studied in in vitro CD3-stimulated cell cultures. In the presence of either IL-2 or IL-15, CD3 stimulation of IEL resulted in low levels of proliferation as measured by thymidine incorporation, whereas no proliferation occurred upon CD3 stimulation in the absence of cytokines. The combination of both cytokines to IEL cultures synergistically enhanced CD3-induced proliferation by approximately threefold that of cultures supplemented with either cytokine alone. Most importantly, however, proliferation of IEL was significantly greater when CD3 stimulation occurred in conjunction with CD43 triggering, indicating that CD43 functions as a coactivational signal for murine IEL. These findings indicate that a spectrum of potential proliferative responses exist among murine IEL depending on the types and combinations of signals received, and that because under normal conditions murine IEL are largely devoid of CD28 expression, a classical T-cell coactivational molecule, the capacity for high-level IEL proliferation may reside with CD43.     

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.     

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs. S7(+) but not S7(-) IELs from the ileum of IL-10(-/-) mice spontaneously produced IFN-gamma. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7(+) population, indicating that significantly more S7(+) IELs than S7(-) IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7(+) IELs expressed higher levels of genes associated with activated T cells, whereas S7(-) IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.     

Glutamine supplementation increases Th1-cytokine responses in murine intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are major effector cells in the gut mucosal immune system, and are phenotypically distinct from thymic and peripheral T cells. Although nutritional supplementation with glutamine affects the intestinal immune response, it remains unclear whether this is a direct effect via the IEL-derived cytokines. This study examined changes in IEL-derived cytokine production following treatment with glutamine in vitro. Murine IELs were purified and activated with PMA plus ionomycin, and then cultured in the presence of various glutamine concentrations. IEL-derived cytokines were measured using a cytometric bead array (CBA) system, and IEL subsets were analyzed by flow cytometry. Treatment with glutamine increased the production of IL-2 and IFN-gamma from IELs in the presence of PMA plus ionomycin, but had no effect on TNFalpha, IL-4, or IL-5 production. Treatment with alanine or glucose had no regulatory effect on IEL-derived cytokines. Glutamine therefore had a direct effect on the production of selected IEL-derived Th1-cytokines, and enteral supplementation with glutamine may influence the intestinal immune responses mediated by IELs.     

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.     

Disturbance of intraepithelial lymphocytes in a murine model of acute intestinal ischemia/reperfusion

Strategically located at the epithelial basolateral surface, intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells and maintain the epithelial barrier integrity. Intestinal ischemia–reperfusion (I/R)-induced acute injury not only damages the epithelium but also affects the mucosal barrier function. Therefore, we hypothesized that I/R-induced mucosal damage would affect IEL phenotype and function. Adult C57BL/6J mice were treated with intestinal I/R or sham. Mice were euthanized at 6 h after I/R, and the small bowel was harvested for histological examination and to calculate the transmembrane resistance. Occludin expression and IEL location were detected through immunohistochemistry. The IEL phenotype, activation, and apoptosis were examined using flow cytometry. Cytokine and anti-apoptosis-associated gene expressions were measured through RT-PCR. Intestinal I/R induced the destruction of epithelial cells and intercellular molecules (occludin), resulting in IEL detachment from the epithelium. I/R also significantly increased the CD8αβ, CD4, and TCRαβ IEL subpopulations and significantly changed IEL-derived cytokine expression. Furthermore, I/R enhanced activation and promoted apoptosis in IELs. I/R-induced acute intestinal mucosal damage significantly affected IEL phenotype and function. These findings provide profound insight into potential IEL-mediated epithelial barrier dysfunction after intestinal I/R.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

Role of murine intestinal intraepithelial lymphocytes and lamina propria lymphocytes against primary and challenge infections with Cryptosporidium parvum

To investigate the role of intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in controlling Cryptosporidium parvum infection, changes in their phenotypes and functional properties were studied after induction of primary and challenge infections in immunocompetent mice. As shown by oocyst-shedding patterns, the challenge-infected group recovered more rapidly from infection than did the primary-infected group. In LPL, proportions of activated CD4+, CD25+, IgG1+, IgA+, and CD4+/IFN-gamma+ cells increased significantly in the primary-infected group compared with controls. In the challenge-infected group, proportions of these cells decreased. The antigen-specific IgA level was elevated significantly among LPL of both primary- and challenge-infected groups. Among IEL, proportions of activated CD8+, T cell receptor (TCR) gammadelta+, and CD8+/TCR gammadelta+ cells increased significantly in the challenge-infected group compared with controls and the primary-infected group; their cytotoxicity also was enhanced. However, the proportion of IEL expressing Th1 cytokines was lower than that among LPL in both infected groups. The results suggest that LPL play a more important role in protection against a primary infection with C. parvum, through the production of IFN-gamma and IgA, whereas IEL are more involved in protection against a challenge infection, through enhanced cytotoxicity.     

Characterization of intraepithelial lymphocytes in human endometrium

Intraepithelial lymphocytes (IELs) were characterized and quantitated in normal non-pregnant endometrium and in early pregnancy decidua using H & E and phloxine tartrazine stains and a panel of monoclonal antibodies in an indirect immunoperoxidase technique. The relative numbers of granulated and non-granulated IELs varied according to menstrual cycle stage and in early pregnancy all IELs appeared to be granulated. There was a higher surface:gland ratio for IELs in proliferative endometrium compared with late secretory phase and early pregnancy endometrium. In proliferative endometrium most IELs were T cells, predominantly of the CD8 + subset. In first trimester decidua, higher numbers of CD56 + cells were observed, in keeping with the increased proportion of granulated IELs. IEL populations in human endometrium vary according to menstrual cycle stage and endometrial IELs appear to show phenotypic differences compared with IELs in the human gastrointestinal tract.     

The light and dark sides of intestinal intraepithelial lymphocytes

The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine form one of the main branches of the immune system. As IELs are located at this critical interface between the core of the body and the outside environment, they must balance protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to do so would compromise homeostasis of the organism. In this Review, we address how the unique development and functions of intestinal IELs allow them to achieve this balance.     

Statins directly suppress cytokine production in murine intraepithelial lymphocytes

Statins, inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are known not only as cholesterol-lowering agents but also as anti-inflammatory mediators. However, their regulatory effect on intestinal mucosal immunity remains unclear. The present study examined the possible direct effects of statin on intestinal intraepithelial lymphocytes (IELs), the front line cells of the intestinal mucosal immune system. Murine IELs were isolated from the small intestines of C57BL/6 mice. IELs activated with anti-CD3/CD28 monoclonal antibodies produced interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 in significant numbers; however, they did not produce IL-5. Both simvastatin and lovastatin suppressed IEL production of IFN-γ, TNF-α, IL-2, and IL-4 in a dose-dependent manner, whereas 48-h treatment with high concentrations (5 × 10^?5 M) of simvastatin and lovastatin did not affect the number of IELs. The suppressive effect of the simvastatin was significantly restored by the addition of mevalonate, farnesyl pyrophosphate ammonium salt, and geranylgeranyl pyrophosphate ammonium salt, which are downstream metabolites of HMG-CoA. These findings suggest that statins have direct suppressive effects on the production of T helper 1-cytokines and IL-4 in IELs; these effects are associated with inhibition of the mevalonate pathway to some extent.     

Oligoclonality of rat intestinal intraepithelial T lymphocytes: overlapping TCR beta-chain repertoires in the CD4 single-positive and CD4/CD8 double-positive subsets

Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR beta-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR beta-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8alphaalpha and CD8alphabeta single-positive (SP) IELs used most Vbeta genes, but relative Vbeta usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vbeta17 and Vbeta19; these two genes accounted collectively for more than half the Vbeta repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping beta-chain repertoires. In conclusion, our results show that rat TCR-alphabeta+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vbeta usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.     

IL-15 promotes survival but not effector function differentiation of CD8+ TCRalphabeta+ intestinal intraepithelial lymphocytes

CD8 single-positive cells, including CD8alphaalpha+ and CD8alphabeta+ subsets, constitute the majority of TCRalphabeta+ intestinal intraepithelial lymphocytes (alphabeta iIEL) in mice. CD8+ alphabeta iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ alphabeta iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ alphabeta iIEL subsets in vitro. In contrast to the death of approximately 60% of both CD8alphaalpha+ and CD8alphabeta+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8alphaalpha+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8alphaalpha+ alphabeta iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15's prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-gamma production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ alphabeta iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8alphaalpha+ alphabeta iIEL upon Ag stimulation.     

Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4+ T cells

CD4+ murine T cell clones, TH1 and TH2, can be distinguished by both functional responses and by their patterns of lymphokine secretion. Recently, a mAb, 23G2, which reacts with a subset of CD45 molecules (CD45R), has been reported to bind differentially to clones of TH1 and TH2 cells. In the present study, normal splenic T cells were analyzed for differences in 23G2-reactivity and were separated into two populations based on their density of CD45R (CD45Rhi and CD45Rlo). The CD45Rhi cells secrete more IL-2 than IL-4 after stimulation in vitro; the reverse is true for the CD45Rlo cells. Because neither population secretes only IL-2 or IL-4, we were unable to classify cells as TH1 or TH2. In vivo and in vitro analyses of the CD45Rhi and CD45Rlo cells suggest a lineage relationship between the two subsets that correlates with the degree of Ag exposure and the state of maturation of the mice. In newborn mice and mice raised under sterile conditions, splenic CD4+ T cells are predominantly CD45Rhi. Under conditions of increased antigenic exposure and maturation of the mice, CD45Rlo cells develop; after long term priming in vivo, the majority of specific Ag-reactive cells are CD45Rlo. Adoptive transfer studies using BALB/c nu/nu recipients demonstrate that CD45Rhi cells become CD45Rlo cells and that the recall response (IgG) to specific Ag is mediated by CD45Rlo cells. Taken together, these data indicate that the level of expression of CD45R on CD4+ T cells distinguishes virgin (CD45Rhi) from primed/memory (CD45Rlo) T cells in normal mice.     

Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

Regulation of cell death and survival in intestinal intraepithelial lymphocytes.

Intraepithelial lymphocytes (IEL) of the small murine bowel represent a unique population of mostly CD8(+) T lymphocytes that reside within the epithelial cell layer of the intestinal mucosa. The close interaction with epithelial cells appears to be crucial for IEL survival since isolation and ex vivo culture induces massive apoptosis in this lymphocyte population. Here, we provide evidence that this form of IEL cell death may be mediated at least in part by endogenously produced glucocorticoids since adrenalectomy or treatment of mice with a glucocorticoid receptor antagonist significantly enhanced ex vivo survival of IEL. We further demonstrate that ex vivo activation of IEL induces upregulation of anti-apoptotic gene products, compensates for the lack of survival cytokines and rescues from apoptotic cell death. Thus, similar to thymocytes and T cell hybridomas, IEL survival may be regulated by the antagonistic action of TCR activation and glucocorticoids.