The Role of αβ⁺ T Cells and Homeostatic T Cell Proliferation in Y-Chromosome-Associated Murine Lupus¹

The primary role of T cells in several spontaneous and induced models of autoimmune syndromes, including lupus, has been clearly demonstrated (reviewed in Ref. 1). With regard to spontaneous models of lupus, the role of T cells in disease pathogenesis has been directly documented in the MRL-Fas^lpr strain. In initial studies, deletion of the TCRα gene, and thus of αβ⁺-expressing T cells, led to a significant, but incomplete, disease amelioration (2). However, disease was almost completely eliminated upon deletion of both TCRα and -δ genes, and thus of αβ⁺ and γδ⁺ T cells (3). For two other spontaneous mouse models of lupus, (NZB × NZW)F₁ and BXSB, studies have only indirectly implicated a role for T cells in disease pathogenesis. In (NZB × NZW)F₁ mice, treatment with Abs to Thy1.2 (4), CD4 (5), MHC class II (6), and B7 (7) or treatment with CTLA4Ig alone (8) or in combination with anti-CD40 ligand (9) resulted in significant disease reduction. Initial efforts with anti-Thy1.2 Ab treatment of BXSB mice were confounded by an unexplained anaphylactic reaction (4), but subsequent experiments with anti-CD4 Ab (10) or CTLA4Ig (11) also reduced disease incidence and severity.

Despite many common characteristics, the three major mouse models of lupus exhibit unique histologic and serologic manifestations as well as unique disease accelerators. These accelerators include female hormones in New Zealand mice, the Fas^lpr mutation in MRL-Fas^lpr mice, and a Y-chromosome-associated accelerator of autoimmunity, termed Yaa,³ in BXSB mice that remains to be identified (reviewed in Ref. 1). Radiation bone marrow chimera experiments with T and B cells of Yaa and non-Yaa origins showed that the determining factor for autoantibody production in BXSB mice was the presence of the Yaa gene in B, but not T cells (12, 13). Other studies have also demonstrated that Yaa primarily increases immune responses to weakly immunogenic Ags (14). Therefore, it was hypothesized that Yaa promotes autoimmunity by enhancing Ag presentation (for example, through increased peptide presentation or increased costimulation), which then facilitates the engagement of otherwise quiescent, low-affinity, self-reactive T cells (12, 13). Further evidence for MHC-dependent T cell engagement in the BXSB disease has been suggested by the inhibitory effects of high I-Eα transgenic expression in this H-2^b mouse (15). This effect appears to be due to competitive inhibition of autoantigen presentation by peptide fragments from processed I-Eα bound effectively to H-2^b class II MHC molecules.

We initially generated congenic TCRα^−/− BXSB mice to directly determine whether αβ⁺ T cells are required in the male BXSB disease and found that such mice were indeed free of disease. We then performed adoptive transfers of small numbers of wild-type (WT) Yaa⁺ or Yaa⁻ mature CD3⁺ T cells to determine whether proliferation of these cells in the lymphopenic Yaa⁺ hosts were equally capable of inducing the full disease spectrum and found that this was the case. Hence, the Yaa gene defect does not modify thymic selection or properties of T cells, but, rather, induces their excessive activation through a non-T cell component.

129/Sv × C57BL/6 TCRα^−/− mice were obtained from M. J. Owen (Imperial Cancer Research Fund, London, U.K.). BXSB TCRα^−/− mice were generated by six backcrosses of the 129 × C57BL/6 TCRα^−/− mice to the BXSB strain, followed by final intercrossing of heterozygous BXSB TCRα^+/− offspring. Control TCRα^+/+ and homozygously deleted TCRα^−/− male BXSB littermates were analyzed in this study. Genotyping for the TCRα deletion was performed by PCR detection of the neo gene, followed by confirmation of the homozygous deletion with anti-CD3 staining and FACS analysis (16). Mice were maintained under specific pathogen-free conditions, and procedures were performed according to guidelines of the institutional animal research committee.

Mutant and control littermates were bled bimonthly and followed for survival until the termination of the experiment. Blood urea nitrogen was measured using AZOSTIX strips (Bayer, Elkhart, IN) according to the manufacturer and was graded on a 1–4 scale (5–90 mg/dl). Histologic examination of periodic-acid Schiff-stained kidneys was performed in a blind manner at 5 mo of age, and the severity of glomerulonephritis (GN) was defined on a scale of 0–4⁺ (16). OCT (Miles, Elkhart, IN)-embedded snap-frozen kidneys were thin sectioned, air dried, fixed in ice-cold acetone, and blocked with 10% horse serum in PBS. Sections were then incubated with anti-IgG-FITC (Vector Laboratories), and deposit intensity was scored as previously described (17).

IgG and autoantibody levels were detected by ELISA as previously described (17). Briefly, IgG in serial dilutions of sera was captured on 96-well plates coated with either the Fc-specific F(ab′)₂ of goat anti-mouse IgG (5 μg/ml; Jackson ImmunoResearch, West Grove, PA) or mouse chromatin (3.5 μg/ml). Bound IgG subclasses were measured using alkaline phosphatase-conjugated goat anti-mouse IgG subclass-specific Abs (Caltag, Burlingame, CA). Standard curves for each subclass were generated using calibrated mouse serum (Binding Site, Birmingham, U.K.).

Splenocytes and PBMC were stained with various combinations of Abs to TCR β-chain, CD3, CD4, CD8, CD19, CD44, and/or CD11b (Mac-1⁺; BD PharMingen, La Jolla, CA). FACS data were acquired (>10,000 events) on a FACSort and were analyzed with CellQuest analysis software (BD Biosciences, Mountain View, CA).

To assess in vivo proliferative responses, two groups (nine mice per group) of αβ⁺ T cell-deficient young male BXSB mice were injected i.v. with 2 × 10⁶ highly purified (95.5% purity) CD4⁺ lymph node (LN) cells from either male or female young BXSB mice (18) that were stained with the intracellular fluorescent dye CFSE (Molecular Probes, Eugene, OR), as previously described (19). Unlabeled donor T cells were analyzed by flow cytometry before injection. Both male and female T cells were <15% CD44^high, <15% CD69⁺, <10% CD25⁺, and >85% CD45RB^high, a phenotype typical of T cells from young unmanipulated BXSB mice. Three mice from each group were sacrificed at 2, 5, and 21 days after transfer, and patterns of cell division as well as total donor T cells (Thy 1.2⁺, CD4⁺) in LN (axillary, inguinal, cervical, mesenteric) and spleen were determined by FACS.

To assess disease induction, young (2-mo-old) αβ⁺ T cell-deficient male BXSB mice were transfused with 4–5 × 10⁶ FACS-sorted CD3⁺ LN cells from 2-mo-old BXSB WT male or female donors (10–12 mice/group) and followed for up to 9 mo of age, and survivors were sacrificed for serologic, cellular, and histologic assessments. A cohort of 20 control WT male BXSB mice was followed in parallel for survival, and an additional 6 were sacrificed at 4 mo for serologic, cellular, and histologic comparisons.

Student’s t test was used for group mean comparisons, and survival was analyzed by the Kaplan-Meier method with comparisons by a log rank test. A value of p < 0.05 was considered to be significant.

To determine to what extent αβ⁺ T cells are required for the development of lupus-like disease in the BXSB model, BXSB mice deficient in the TCR α-chain were generated, and the severity of autoimmune manifestations in male (Yaa⁺) TCRα^−/− and littermate TCRα^+/+ controls was compared. The absence of detectable αβ⁺ T cells in the TCRα^−/− mice was confirmed by flow cytometry using Abs to the TCR β-chain. Mice were followed for survival for 9 mo, well beyond the life expectancy of BXSB males (1). Strikingly, the lack of TCRαβ⁺ cells resulted in a dramatic increase in survival (Fig. 1). Control BXSB males exhibited 50% mortality at 5.3 mo and 100% mortality by 6.5 mo, while BXSB TCRα^−/− male mice were all alive at 9 mo (p < 0.0001). Notably, the mortality rate of the littermate controls was similar to that of our WT BXSB colony, indicating that the congenic line contained the major BXSB lupus susceptibility genes.

Serum polyclonal IgG levels were determined in TCRα^−/− and TCRα^+/+ littermates at 5 mo of age. Although the controls exhibited typical hypergammaglobulinemia, all IgG subclasses were significantly lower in TCRα^−/− mice (p < 0.001), resulting in a >9-fold reduction of total serum IgG (Fig. 2,A). Nonetheless, the IgG levels and subclass distributions in these mutant mice were similar to those commonly found in normal genetic background mice, an observation consistent with reports indicating considerable class switching in the absence of αβ⁺ T cells (20). Our previous studies have shown that the autoantibody response to native chromatin in lupus-predisposed strains, including BXSB, generally preceded the appearance of autoantibodies against chromatin subcomponents, such as dsDNA and histones (21). To evaluate disease progression in the groups studied, we determined antichromatin titers at 5 mo of age and detected high levels, predominantly of the IgG2a subclass, in TCRα^+/+ littermates, while such autoantibodies were virtually absent in the deficient mice (Fig. 2 B).

Weights and cellular composition of spleen and lymph nodes were also analyzed (Table I). As expected, there was a significant reduction in the weights of both organs in the TCRα^−/− mice compared with controls (p < 0.05) with, on the average, a 10-fold decrease in LN and a 2-fold decrease in spleens. Similarly, deficient mice were devoid of T cells, while, as previously reported (22, 23), the controls showed a large proportion of CD44^high activated/memory phenotype T cells (Table I). In TCRα^−/− mice, however, there was an incremental increase in the frequency of CD19⁺ B cells, probably due to the absence of αβ⁺ T cells.

Previous studies have shown the accumulation of a peculiar Mac-1⁺ MHC class II⁻ monocyte population in the peripheral blood of male, but not female, BXSB mice (24). To determine whether this monocytosis is T dependent, we analyzed Mac-1⁺ expression in PBMC of 5-mo-old mice (Table I). Although control TCRα^+/+ mice exhibited the typical expansions of Mac-1⁺-expressing cells (21.2%), this population was significantly reduced (8.9%) in TCRα^−/− mice (p < 0.01). The reduction in absolute numbers of these cells is probably more severe than the indicated drop in percentage suggests, since αβ⁺ T cells are not present in the deficient mice.

Kidney weights, GN scores, and blood urea nitrogen levels were reduced (p < 0.05) in 5-mo-old BXSB TCRα^−/− mice compared with TCRα^+/+ controls (Table II). Control BXSB male mice exhibited typical glomerular pathology, including extended glomeruli, sclerosis of glomerular capillary walls, and heavy periodic acid-Schiff-positive material in the mesangial matrix (Fig. 3). Hypercellularity of the mesangium and the presence of both mononuclear and polymorphonuclear cells were also noted in the control mice, as were heavy glomerular IgG immune deposits in the mesangium and capillary walls. All these parameters were considerably reduced in TCRα^−/− mice (Fig. 3).

Several recent studies have shown that small numbers of T cells transferred into lymphopenic hosts proliferate extensively to reconstitute the original lymphocyte pool (reviewed in Refs. 25, 26, 27). This proliferation appears to be mediated by recognition of self-MHC/peptide ligands. Therefore, male mice deficient in αβ⁺ T cells, and thus lymphopenic, were adoptively transferred with small numbers of CFSE-stained CD4⁺ LN cells from WT male and female donors to determine the degree of homeostatic proliferation. In agreement with previous studies (18), no proliferation was detected on day 2 after transfer, since CFSE-stained cells were confined to a single peak on the far right of the histogram (data not shown). Thereafter, both WT male and female CD4⁺ T cells transferred into αβ⁺ T cell-deficient male mice proliferated extensively and equally, thereby leading to reduced CFSE intensity in 87–89% of donor cells on day 5 and 96% on day 21 after transfer (Fig. 4,A). Consequently, enumeration of donor T cells (Thy 1.2⁺, CD4⁺) at these two time points showed a nearly identical recovery regardless of donor gender (Fig. 4 B).

Since αβ⁺ T cell-deficient mice were free of disease and mature T cells transferred into lymphopenic hosts homeostatically expanded, we performed adoptive transfers of small numbers (4–5 × 10⁶) of WT male or female CD3⁺ LN cells into male αβ⁺ T cell-deficient mice to determine whether T cell origin affects disease reconstitution in the lymphopenic male BXSB background.

Male recipients of either male or female T cells exhibited equal mortality rates, with ∼50% of the animals dead at 9 mo of age, or 7 mo after transfer, the latest point of observation (Fig. 5). This mortality rate is delayed ∼2 mo compared with an unmanipulated cohort of control WT male BXSB mice, which may be explained by the time required for the small number of transfused cells to expand sufficiently. Serologic analyses showed that increases in polyclonal IgG (Fig. 6,A) and antichromatin (Fig. 6,B) subclasses of male αβ⁺ T cell-deficient recipients of either male or female WT T cells were equal and approximated those of the unmanipulated WT mice. Similarly, spleen and LN weights and cellular compositions, including high percentages of activated/memory phenotype CD4⁺CD44^high and CD8⁺CD44^high cells were equal in the three groups, as was the frequency of Mac-1⁺ monocytes in peripheral blood (Table III). Finally, blood urea nitrogen levels, GN severity, and intensity of IgG kidney deposits were also very similar between the T cell-reconstituted and control WT mice regardless of the gender origin of T cells (Table IV). Overall, the results indicate that homeostatically proliferating mature T cells of male or female BXSB origin are equally capable of inducing an early life, lupus disease in the αβ⁺ T cell-deficient male BXSB background.

We have conclusively demonstrated, through analysis of congenic TCRα-deleted BXSB mice, that αβ⁺ T cells are necessary for lupus expression in this strain. The αβ⁺ T cell-deficient male mice showed no mortality after 9 mo and had severely reduced serum IgG and antichromatin autoantibody levels, decreased peripheral blood monocytosis, and minor grades of GN. The full spectrum of disease could, however, be reconstituted in these male T cell-deficient mice by both Yaa⁺ (male BXSB) and Yaa⁻ (female BXSB) T cells, thereby documenting that homeostatic anti-self T cell proliferation can induce systemic autoimmunity in an appropriate background, and that the Yaa gene functions by impacting a non-T cell component.

It is of considerable interest that the absence of αβ⁺ T cells led to an almost complete elimination of disease in the BXSB males. This effect was more pronounced than that observed by Craft and associates (2) in similarly TCRα-deleted MRL-Fas^lpr mice in which only partial protection from disease was afforded. In this strain concurrent deletion of both the α and δ genes, and thus of both αβ⁺ and γδ⁺ T cells, was required for nearly complete elimination of serologic and histologic disease parameters (3). The present findings in the BXSB mouse are also at some variance with the initial studies by Wen et al. (28), who reported that αβ⁺ T cell-deficient normal background mice showed expansion of B cells and secretion of T-dependent isotype autoantibodies (IgG1 and IgE) with a spectrum of specificities similar to those in lupus, suggesting that autoantibody induction can be mediated by γδ⁺ T cells. Adoptive transfer experiments into SCID mice by these investigators indeed showed that γδ⁺ T cells are capable of providing help to B cells in the absence of αβ⁺ T cells (20). It appears, therefore, that the ability of γδ⁺ T cells to promote systemic autoimmunity is dependent upon additional genetic and/or environmental factors. Recent studies have shown that CD4⁺ αβ⁺ T cells of MRL-Fas^lpr mice are hyper-responsive to antigenic stimuli (29). Such enhanced responses may also be applicable to MRL-Fas^lpr, but not to BXSB, γδ⁺ T cells.

A unique characteristic of the BXSB male disease, originally identified by Wofsy et al. (24), is the appearance of a late-onset monocytosis, almost exclusively detected in the peripheral blood. These cells appear atypical in that despite expressing Mac-1 and having morphological features of macrophages, they are devoid of MHC class II molecules. The origin and potential contribution of these cells to the disease process remain unclear. The present study as well as a previous study in which monocytosis was reduced in BXSB male mice treated with anti-CD4 Ab (10) document that this peculiar manifestation is highly T cell dependent. The means by which T cells induce mobilization of these monocyte-like cells remains unknown, but several products of activated T cells, including IFN-γ (30), M-CSF (30), and GM-CSF (31), might be the mediators. It is apparent, however, that simple activation of T cells and secretion of monocytosis-promoting products cannot fully account for this manifestation, since large numbers of activated T cells are also present in other lupus strains of mice that do not exhibit this characteristic. It is possible, therefore, that induction of the putative T cell-derived factor(s) is directly or indirectly connected to the presence of the Yaa gene. The picture becomes more complicated, however, if one considers that this monocytosis does not occur in a long-lived subline of BXSB male mice previously established in this laboratory (32). Because appropriate breeding experiments showed that longevity in this BXSB subline was not due to a modification of the Yaa gene, it should be concluded that the monocytosis-promoting factor(s) is an intermediary between T cells and the Yaa gene product. Further studies on the means of induction, homing patterns, marker acquisition, and functional characteristics of these monocyte-like cells are, therefore, highly warranted.

The mode by which the Yaa gene defect accelerates a lupus-like disease in appropriate backgrounds has not been fully elucidated, and the actual gene remains unknown. Nevertheless, Izui and associates (12, 13, 14) showed that in bone marrow chimeras containing two sets of T and B cells from mice with or without the Yaa gene, the T cells from either Yaa⁻ nonautoimmune mice or Yaa⁺ autoimmune mice were equally efficient in promoting anti-DNA and anti-gp70 autoantibody production by Yaa⁺ B cells. Therefore, they concluded that the Yaa gene defect is not functionally expressed in T cells, but only in B cells (and/or other APCs), and suggested that this defect leads to the engagement of otherwise quiescent, low avidity, self-reactive T cells. Indeed, recent studies have shown that normal mice harbor a large cohort of these normally harmless cells, which, under certain circumstances, such as high peptide presentation and costimulation, may acquire effector function and become harmful (33).

Our findings with adoptive transfers of Yaa⁺ and Yaa⁻ BXSB mature T cells into the αβ⁺ T cell-deficient BXSB male mice are in full congruence with the above conclusions and further extend the results of Izui and associates (12, 13, 14) in demonstrating full recapitulation of the male-like BXSB disease with either type of T cell. Adoptive transfer experiments with mature T cells became feasible through the availability of the lymphopenic (i.e., TCRα^−/−) BXSB mice, wherein transferred T cells survived and homeostatically expanded to reconstitute the normal lymphocyte pool. Numerous recent studies have documented that homeostatic T cell proliferation in the periphery is based on recognition of self-MHC/peptide ligands similar to those used in intrathymic positive selection, and the expanded cells acquire activation markers as well as effector function (25, 26, 27). Of interest, however, only a small fraction (∼15%) of mature T cells are expected to engraft and proliferate in a lymphopenic host (34). Why only a small fraction exhibits this capacity is unclear, but likely possibilities include the absence or low expression levels in the periphery of some of the positive selection-mediating thymic peptides, restriction of proliferative capacity to T cells with higher anti-self avidity, and/or cell death. Accordingly, skewing of the Vβ TCR repertoire and a reduction in humoral responses to diverse Ags following homeostatic expansion of polyclonal T cell populations have been observed (35). In our transfers of 4–5 × 10⁶ mature T cells, it can therefore be calculated that reconstitution of the T cell pool occurred via the expansion of as few as 6–7 × 10⁵ (∼15%) T cells. These few cells, likely to encompass a small repertoire of TCRs, were, nevertheless, sufficient to induce the full serologic and histologic spectrum of the male BXSB disease. In future studies, we will determine the minimum number of T cells required to induce disease via homeostatic expansion in this setting. These issues aside, it can be hypothesized that anti-self-homeostatic expansion of T cells in appropriate backgrounds may play a primary or secondary role in the initiation and perpetuation of systemic autoimmunity, and the findings presented herein provide initial evidence that this might be the case.

We thank C. B. Wilson and S. T. Duncan for advice, M. J. Owen for the initial breeding pair of 129/Sv × C57BL/6 TCRα^−/− mice, D. Balomenos for initial mouse breeding, and M. Kat Occhipinti-Bender for editorial help.