Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4+ effector cells that are resistant to regulatory T cell activity

In Fas/FasL-deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3(+) Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220(-) CD4(+) T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-gamma differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.     

Increased Ia expression, T lymphocyte subset abnormalities and autoimmunity in murine strains bearing the lpr gene.

Defects in cellular communication are fundamental to the development of autoimmune disease. Modulation of immunoregulatory events can be mediated by cellular expression of Ia antigens. We have analysed, by flow cytometry, the Ia antigenic levels on cells from mice expressing the lpr gene and their congenic counterparts. Surface Ia expression is dramatically increased on bone marrow, thymus, lymph node and spleen cells from lpr mice even prior to characteristic lymph node and spleen enlargement. In addition, IL-2 production abnormalities occur in the low density Lyt 1 subset of Thy 1.2 positive cells of normal mice which may be the counterpart of the majority cell type of lpr lymphocytes. Treatment of lpr mice with low dose whole body irradiation (300 rad) decreases lymphadenopathy, autoantibodies, proteinuria and the resident Ia positive cell population while increasing survival. We conclude that lymphoid alterations induced by irradiation reflect a recovery of immunological control associated with suppression of autoimmune manifestations.     

T lymphocyte colonies are the progeny of single cells.

T lymphocyte colonies, arising from phytohemagglutinin (PHA) stimulated mononuclear cells cultured in a semi-solid agar matrix, could be the progeny of single cells (monoclonal) or of multiple cells (polyclonal). We have conducted several studies to determine if these colonies are monoclonal or polyclonal in origin. Normal human peripheral blood mononuclear cells from male-female, HLA-A and B disparate donor pairs were incubated for 18 h in RPMI 1640 containing PHA and fetal calf serum (FCS) and then cultured in a two-layer semi-solid agar system. After 5 days of incubation, the clonality of the colonies was assessed by in situ Y chromatin analysis, and by analysis of HLA-A and B locus antigens. Overlayers were stained with quinicrine dihydrochloride and the number of cells in the T cell colonies with Y chromatin enumerated using fluorescence microscopy. In other studies, colonies were picked from the agar with a capillary pipette and expanded in culture media. After 17 days of culture, cells were harvested and HLA-A and B phenotypes were determined. The results indicate that 87% of the T cell colonies had cells of either male or female origin. In addition, 90% of the colonies possessed HLA-phenotypes of only one donor. We conclude that Y chromatin and HLA analysis of individual colonies from cocultures suggest the monoclonality of T lymphocyte colonies.     

lpr T cells vaccinate against lupus in MRL/lpr mice.

MRL/MP-lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8-) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease-accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp-lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This "T cell vaccination" approach resulted in a selective depletion of cells expressing products of the V beta 8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.     

The lymphoproliferating cells of MRL-lpr/lpr mice are a polyclonal population that bear the T lymphocyte receptor for antigen

Mice bearing the recessive gene lpr develop an age-dependent, massive lymphoproliferation, primarily in the lymph nodes (LN), with associated autoimmunity. LN cells from these mice express T cell receptor protein on the cell surface at 50-70% of normal levels. Normal levels of T cell receptor alpha, beta and gamma mRNA were found in these cells as compared to normal LN cells. Southern blot analysis of MRL-lpr/lpr LN DNA showed rearrangements in 80-90% of the chromosomes at the beta gene loci. The pattern of rearrangement indicated that a polyclonal rather than monoclonal expansion of T cells occurred. These data support a lymphokine-induction model of lymphoproliferation in MRL-lpr mice.     

Unusual cell surface properties of the T lymphocyte population expanding in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (lpr/lpr) mice but not the congenic MRL/Mp-+/+ (+/+) mice, develop a generalized lymph node (LN) hypertrophy reflecting the expansion of a T-cell population that acts as an enhancing factor for autoimmunity. In order to characterize better this T-cell population, we investigated some of its surface properties in comparison with those of +/+ T cells. Electrophoretic measurements revealed that lpr/lpr T cells possess a lower electronegative surface charge than %/% T cells which indicates that the two cell types differ in the molecular composition of their plasmic membrane periphery. This notion was substantiated by the quantification of T- and B-cell markers and of lectin-binding sites on these cells using single- and two-colour flow cytofluorimetry. In agreement with recent observations by Lewis, Giorgi & Warner (1981) lpr/lpr T cells exhibited lower levels of Thy-1 and Lyt-1 antigens than +/+ T cells and were mostly devoid of Lyt-2 antigen. Although lpr/lpr lymph node (LN) cells displayed similar amounts of surface receptors for peanut agglutinin as +/+ LN cells, the expression of surface receptors for other lectins were either lower (Limulus polyphemus agglutinin, Maclura pomifera agglutinin, Concanavalin A) or higher (Helix pomatia agglutinin, Soya bean agglutinin, Bandeiraea simplicifolia agglutinin I, Phytohaemagglutinin L) on lpr/lpr T cells than on +/+ T cells. These data indicate that the T cells accumulating in hypertrophied lpr/lpr LN are endowed with unique surface characteristics which may explain some of the functional abnormalities of these cells.     

5-Azacytidine inhibits the lpr gene-induced lymphadenopathy and acceleration of lupus-like syndrome in MRL/MpJ-lpr/lpr mice.

MRL/MpJ-lpr/lpr mice spontaneously develop a lupus-like autoimmune disorder characterized by massive proliferation of T cells and rapidly fatal immune complex glomerulonephritis. We evaluated the therapeutic effect of 5-azacytidine (5AC), a cytidine analogue known as an inhibitor of DNA methylation, in MRL/MpJ-lpr/lpr mice. Intraperitoneal injection of 5AC (50 micrograms, twice a week) starting from 6 weeks of age retarded the development of lymphadenopathy and autoimmune syndrome. Its beneficial effects included: (a) increased life-span, (b) diminution of lymphadenopathy and splenomegaly, (c) reduction in circulating levels of autoantibodies such as anti-DNA and rheumatoid factors, and (d) suppression of lupus glomerulonephritis. However, similar treatment in BALB/c mice did not affect the development of IgG anti-human IgG antibody responses. These results suggest that the protective effect of 5AC is related to the inhibition of the lpr gene-induced T cell proliferation, thereby suppressing the autoimmunity-accelerating effect mediated by the lpr gene.     

T cell receptor-bearing cells among rat intestinal intraepithelial lymphocytes are mainly alpha/beta+ and are thymus dependent

Rearrangement of both the beta and gamma chain T cell receptor (TcR) genes was detected in intestinal intraepithelial lymphocytes (IEL) from normal euthymic rats. Flow cytometric analyses showed that about 73% of the IEL were CD3+ (1F4) and that 67% were TcR alpha/beta+ (R73). About 5% of the IEL were found to be CD3+, TcR alpha/beta- in double-labeling experiments suggesting that a small fraction of IEL in the rat express the alternative TcR gamma/delta. More than 70% of the IEL were granular implying that many CD3+ IEL are granular. In IEL from athymic nude rats no rearrangement of either the TcR beta or gamma chain genes or surface expression of CD3 or TcR alpha/beta was detected despite the fact that about 95% of the cells were granular and morphologically similar to those in normal rats. Taken together our data suggest that the majority of IEL in the rat express the conventional TcR alpha/beta and that TcR-bearing cells in the gut epithelium are thymus dependent.     

Mechanisms of peripheral T cell deletion: anergized T cells are Fas resistant but undergo proliferation-associated apoptosis

The complementary receptor pair Fas ligand: Fas controls apoptosis during activation-induced cell death (AICD) of peripheral T cells sensitized for the Fas signal pathway by interleukin-2 (IL-2). In the present study, we used the bacterial superantigen staphylococcal enterotoxin B (SEB) to anergize ligand-reactive peripheral T cells in wild-type and Fas-defective lpr mice. In a second step, we investigated whether apoptosis in anergized and thus operationally IL-2-defective peripheral T cells is triggered via the Fas signal pathway. We report here that SEB-driven anergy induction and deletion of anergized peripheral Vβ8⁺ T cells is similar in wild-type and healthy C3H/lpr mice. In monitoring SEB-driven Vβ8⁺ T cell apoptosis in situ, we observe in both wild-type and lpr mice an intimate association between proliferation and apoptosis of anergized Vβ8⁺ T cells. We further show that Vβ8⁺ T cells activated in vitro from wild-type mice express a Fas-sensitive phenotype determined by Fas cross-linking which causes apoptosis. In contrast, Vβ8⁺ T cells anergized in vivo from wild-type mice are Fas resistant. As expected, T cells from lpr mice activated in vitro or anergized in vivo are Fas resistant. Taken together, these data indicate that both in wild-type and Fas-defective C3H/lpr mice, anergized T cells become deleted via a Fas-independent, proliferation-associated apoptosis signal pathway.     

IL-12 enhances lymphoaccumulation by suppressing cell death of T cells in MRL- lpr/lpr mice

Lymphoaccumulation occurs in MRL- lpr/lpr mice, because double-negative T cells (DNT cells) cannot be deleted due to their Fas mutation, i.e., lpr. We show here that IL-12 enhances in lymphoaccumulation by suppressing cell death of DNT cells in [corrected] MRL- lpr/lpr mice. It has been reported that viable DNT cells from MRL- lpr/lpr mice undergo rapid apoptosis in ordinary cell culture without additional stimulation, suggesting that unknown in vivo factors other than lpr suppress the apoptosis. In the present study, we found that plasma IL-12p40 monomer and/or homodimer level increased with age in MRL- lpr/lpr but not in MRL-+/+ mice, and the increase in IL-12 correlated well with lymphoaccumulation. Requirement of IL-12 in lymphoaccumulation and in suppressed cell death of DNT cells of MRL- lpr/lpr mice was assessed. When an antibody neutralizing IL-12 was injected into old MRL- lpr/lpr mice with high plasma IL-12 level, lymphoaccumulation was diminished. When IL-12p40- or IL-12p70-encoding plasmid was administered to young MRL- lpr/lpr mice before the plasma IL-12 level increases, lymphoaccumulation was enhanced. The ordinary cell culture-induced cell death of DNT cells from MRL- lpr/lpr mice was suppressed in the presence of IL-12. Since DNT cells produce IFN-gamma, a potent inducer of IL-12, the INF-gamma induced-IL-12 may enhance lymphoaccumulation in MRL- lpr/lpr mice.     

Antigen-specific helper T cells are essential for cytotoxic T cell responses to metabolically inactivated stimulator cells.

Primed spleen cells respond well to metabolically inactivated stimulator cells while normal spleen cells do not. This observation has been interpreted as showing that cytotoxic T cell precursors are different from unprimed precursors in their antigen recognition requirements for induction. A different model is proposed here which accounts for these observations as due to enhanced helper cell levels in primed populations. Experiments are described in this study which test several predictions of this model. These experiments show that in the presence of in vitro primed helper T cells, normal cells are able to respond efficiently to glutaraldehyde-fixed stimulator cells. The helper effect is antigen-specific. Since unprimed spleen cells can be efficiently induced by metabolically active stimulators (γ-irradiated cells) and can respond to glutaraldehyde-fixed antigen (metabolically inactive cells) only in the presence of specific helper cells, it seems reasonable to propose that helper cell signals are enhanced by a nonantigenic property of γ-irradiated stimulator cells requiring metabolic activity. It is also clear that glutaraldehyde-fixed cells are anti- genically intact as helper cells, primed to antigens on γ-irradiated stimulator cells, efficiently and specifically help a response to fixed stimulators. Conversely, helper cells primed in vitro to glutaraldehyde-fixed stimulators recognize antigen on γ-irradiated stimulator cells. The level of help generated in response to glutaraldehyde- fixed stimulator cells is at least 10-fold higher in primed cells than in normal cells. In addition, primed spleen cells can be induced in vitro to yield helper function by both fixed or unfixed stimulator cells. Normal helper cell precursors are induced at least 100-fold more efficiently by γ-irradiated as compared to glutaraldehyde-fixed stimulator cells. This work supports the idea that a major effect of priming, which allows primed cells to respond to metabolically inactive stimulators, is to enhance levels of helper T cells in the primed population.     

Lymphopenia-driven CD8(+) T cells are resistant to antigen-induced tolerance in NOD.scid mice

T cells undergoing lymphopenia-driven proliferation acquire effector and memory properties that can be pathogenic. Indeed, generalized lymphopenia is associated with a variety of autoimmune diseases such as type 1 diabetes. The current study was carried out to determine how CD8(+) T cells undergoing acute lymphopenic expansion respond to antigen under tolerizing conditions in vivo. Adoptive transfer of diabetes by TCR-transgenic CD8(+) T cells was enhanced following treatment of NOD. scid recipients with a high dose of soluble peptide. Furthermore, whereas TCR-transgenic CD8(+) T cells underwent clonal deletion and failed to differentiate into CTL in peptide-treated lymphoreplete recipient mice, TCR-transgenic CD8(+) T cells in a lymphopenic environment were resistant to clonal deletion, and CTL differentiation was enhanced by a high dose of soluble peptide. Moreover, peptide treatment had distinct effects on expression of the anti-apoptotic protein Bcl-X(L) in TCR-transgenic CD8(+) T cells under lymphopenic versus lymphoreplete conditions. These results demonstrate that CD8(+) T cells undergoing lymphopenia-driven expansion in NOD. scid recipients are resistant to antigen-induced tolerance, and readily differentiate into CTL upon stimulation with a high dose of soluble peptide.     

Changing jejunal gamma delta T cell receptor (TCR)-bearing intraepithelial lymphocyte density in coeliac disease.

The function of jejunal intraepithelial gamma delta+ T cells is obscure, but they are commonly implicated as playing a role in inflammatory and autoimmune conditions. In coeliac disease (CoD), there are controversial reports as to gluten dependency of these cells. We have now studied the small bowel mucosal intraepithelial T cell densities, and the ratios of gamma delta+ to CD3+ T cells and gamma delta+ to alpha beta+ T cells during early disease development and on a gluten-free diet. Nine children initially excluded for CoD were followed up and rebiopsy after 0.8-4.5 years showed mucosal deterioration. Further, 21 biopsy specimens from newly diagnosed CoD patients were studied, together with 20 specimens taken from children on a gluten-free diet. During CoD development the density of gamma delta+ and alpha beta+ T cells as well as the ratios of gamma delta+ to CD3+ T cells and gamma delta+ to alpha beta+ T cells increased. In the latent stage of CoD when the small bowel mucosal architecture was still normal, two children had clearly normal densities of gamma delta+ (< 2.5 cells/100 epithelial cells) and alpha beta+ (< 25.0 cells/100 epithelial cells) T cells, and low ratios as well. In patients with newly diagnosed CoD the densities decreased significantly on a long-term gluten-free diet. We conclude that the density of intraepithelial gamma delta+ T cells as well as alphabeta+ T cells in CoD is gluten-dependent. CoD can develop in a child ingesting normal amounts of gluten and having normal jejunal mucosal morphology on biopsy and a normal density of gamma delta+ T cells.     

Clonal analysis of peripheral T cell precursors in lpr mice.

MRL/Mp-lpr/lpr mice develop massive lymphadenopathy characterized by expansion of an unusual population of T cells with the Thy 1+, CD3+, CD4-, CD8- (double negative) phenotype. The role these cells play in accelerating the autoimmune syndrome seen in these mice is unknown. In order to better understand the origin of the expanded population of T cells, we have derived a panel hybridomas from double negative lpr lymph node cells. Surprisingly, eleven of twelve hybridomas selected for the absence of surface CD4 and CD8 do not express CD3. Six of eleven confirmed to have inherited the MRL T cell receptor locus have rearrangement at that locus, suggesting commitment to a T cell lineage. Only hybridoma 2.4, which expresses CD3, responds to ConA, anti-CD3 monoclonal antibody, and induces antibody production. The presence of CD3-, CD4-, CD8- T cells in the periphery of lpr mice confirms aberrant T cell development in these mice and suggests an intrinsic cell defect which is expressed early in lymphopoiesis.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.     

Intestinal intraepithelial and splenic natural killer cell responses to eimerian infections in inbred chickens.

Splenic and intestinal natural killer (NK) cell responses were assessed in chickens inoculated with Eimeria parasites. The NK cell activities of both splenic and intestinal intraepithelial lymphocytes (IELs) decreased to a subnormal level during the early phase of eimerian infections but returned to normal or slightly higher than normal levels at about 1 week after the primary inoculation. Lymphocytes obtained from the lamina propria did not show any detectable level of NK cell activity during or following eimerian infections. Significant increases in splenic and intestinal IEL NK cell activities were seen during the early phase of secondary infection. The increase in the IEL NK cell activity that was seen shortly following secondary eimerian infection was accompanied by a substantial increase in the number of IELs expressing the asialo-GM1 antigen. Host strain differences in both splenic and IEL NK cell responses were detected following primary eimerian infections. These results suggest that both splenic and intestinal IEL NK cells may play an important role in the host defense against intestinal protozoan infections.