An increased frequency of autoantibody-inducing CD4+ T cells in pre-diseased lupus-prone mice

Brian W. Busser1, Michael P. Cancro2 and Terri M. Laufer1

1 Department of Medicine and 2 Department of Pathology and Laboratory Medicine University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence to: T. M. Laufer; E-mail: tlaufer{at}mail.med.upenn.edu
Transmitting editor: L. H. Glimcher


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pathogenic autoantibody production in murine models of lupus is dependent on autoreactive CD4+ helper T cells. However, the mechanisms which permit the selection and maintenance of this autoantibody-inducing CD4+ T-cell repertoire are currently unknown. We hypothesized that the peripheral CD4+ T-cell repertoire of lupus-prone mice was enriched with autoantibody-inducing specificities. To test this, we utilized the splenic focus assay to determine if pre-diseased lupus-prone (NZB x NZW)F1 mice have an elevated frequency of autoreactive CD4+ T lymphocytes capable of supporting autoantibody production. The splenic focus limiting dilution assay permits anti-nuclear antibodies to be generated from contact-dependent T–B interactions in vitro. We show that young, pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T cells. Interestingly, these autoantibody-inducing CD4+ T-cell responses are also present in the thymus. Therefore, an elevated frequency of autoantibody-inducing CD4+ T cells predisposes lupus-prone mice to the development of autoantibodies.

Keywords: autoantibodies, B lymphocytes, lupus, T lymphocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The activation of autoreactive T lymphocytes leads to both systemic and organ-specific autoimmunity. The inappropriate activation of organ-specific autoreactive CD4+ T cells underlies insulin-dependent diabetes mellitus and multiple sclerosis; autoreactive T lymphocytes also engender the autoantibody production characteristic of SLE. Several inbred mouse strains are genetically predisposed to autoimmunity (13), but the relationship of these genetic factors to the selection and activation of self-reactive T cells remains unclear. For example, an increased frequency of proteolipid protein (PLP)-reactive T cells is present in the experimental allergic encephalomyelitis (EAE)-susceptible SJL strain, reflecting both aberrant thymic selection and peripheral homeostasis (4,5). Similarly, diabetes-prone NOD mice may have a thymic defect which predisposes to the accumulation of autoreactive T cells (6). Thus, predisposition to the development of organ-specific autoimmunity includes an increased frequency of autoreactive CD4+ T cells. Whether this characteristic is shared by individuals prone to systemic autoimmune diseases such as lupus remains to be determined.

In this study, we describe an in vitro experimental system which allowed us to estimate the number of CD4+ T cells in a particular repertoire capable of driving autoantibody production in a normal splenic micro-environment. We utilized the splenic focus assay, a limiting dilution strategy originally developed to study individual B cell or CD4+ T helper cell responses to nominal antigens in vitro (7,8). The assay relies on supplying a splenic environment in which limiting numbers of CD4+ T cells induce B cell production of antibodies in vitro and determines the functional precursor frequency of the limiting input cell type. We find that the precursor frequency of autoantibody-inducing CD4+ T cells in pre-diseased, lupus-prone (NZB x NZW)F1 (BWF1) mice is significantly higher than that of non-autoimmune mice. Interestingly, the autoantibody-inducing CD4+ T cells are also present in the thymus; a high frequency of CD4 single-positive (SP) thymocytes from BWF1 mice could help B cell production of anti-chromatin autoantibodies. Thus, mice prone to both organ-specific and systemic autoimmunity have T-cell repertoires enriched with autoreactive T cells which contribute to the development of disease.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
BWF1, MRL/MpJ-Faslpr (MRL-lpr), H-2bm12 and C57Bl/6J (B6) mice were purchased from Jackson Laboratories and maintained in a specific pathogen-free environment at the University of Pennsylvania Medical Center. All mice were used between the ages of 4–6 weeks.

CD4+ T-cell isolation
All visible lymph nodes were removed from donor mice and mashed between the frosted ends of glass slides to generate single cell suspensions. CD4+ T cells were isolated using a negative selection magnetic sorting approach as previously described (9,10). In brief, single cell suspensions were incubated with pre-titrated volumes of anti-CD8 (2.43), -B220 (RA3-B220), -FcR (2.4G2) and -MHC class II (M5/114) hybridoma tissue culture supernatants and non-CD4+ T cells were removed with magnetic bead-conjugated goat anti-rat Ig (Polysciences, Inc.) attached to a Bio-Mag magnetic stand (Polysciences, Inc.). CD4+ T-cell purity was >90% when checked by flow cytometry.

CD4 SP thymocyte isolation
Thymi from donor mice were mashed with glass slides to generate a single cell suspension. CD4 SP thymocytes were isolated in a similar manner as peripheral CD4+ T cells except thymocytes were only incubated with anti-CD8 (2.43) supernatant. CD8+ cells were removed with magnetic bead- conjugated goat anti-rat Ig attached to a magnetic stand. CD4 SP thymocyte purity was >80% when checked by flow cytometry.

B cell isolation
Spleens from donor mice were mashed with glass slides to generate single cell suspensions. Splenocytes were incubated with anti-Thy1.2 (MMT1), anti-CD4 (172-4) and anti-CD8 (TIB-211) for 45 min on ice. Rabbit complement (Cedarlane Laboratories, Ltd) was then added for 30 min (37°C) and cells were washed. B cell purity was >90% when checked by flow cytometry.

Limiting dilution splenic focus assay
The limiting dilution splenic focus assay was modified from the original protocol developed by Froscher and Klinman (8). Recipient mice were {gamma}-irradiated (1500 rads) 4 h prior to being injected i.v. with titrated doses of CD4+ T cells and 50 x 106 B cells in Hank’s Balanced Salt Solution. The non-limiting dose of B cells was established by titrating increasing numbers of B cells into the splenic focus assay with a non-limiting dose of CD4+ T cells—the plateau of the B cell response occurs at a non-limiting dose. Recipients spleens were removed 18 h later, chopped into 1 mm3 fragments (Tissue-Tek tissue chopper), and individually plated in flat bottom 96-well plates in cell culture DMEM medium at 37°C, 92% O2, 8% CO2. Medium was replaced every 3 days; culture supernatants were collected beginning on day 9 and frozen at –20°C until assayed by ELISA for reactivity to chromatin. Fragment cultures were scored positive for anti-chromatin antibodies when the O.D. was 2 standard deviations greater than the mean of fragment cultures which received B cells alone (10,11). For some cultures, anti-CD40L [clone MR1; 3 µg/ml (12)] or control hamster IgG (Sigma Chemical Co.; 3 µg/ml) was added to culture media. The precursor frequency of autoantibody-inducing CD4+ T cells was determined by linear regression according to previously published methods and was corrected for homing efficiency of CD4+ T cells (13). Homing efficiency is the percentage of injected CD4+ T cells which migrate to the spleen 18 h after injection, and was quantified using CFSE labeling of the input CD4+ T cells.

Anti-chromatin ELISAs
Culture supernatants or a 1:500 dilution of serum were added to bovine thymus chromatin-coated ELISA plates and anti-chromatin IgG was detected as previously described (10,11).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Limiting dilution splenic focus technique
Autoantibody production in lupus-prone mice is dependent on CD4+ T-cell help (1416). We utilized the limiting dilution splenic focus assay to determine whether an increased frequency of autoreactive CD4+ T cells precedes the development of anti-chromatin antibodies in lupus-prone mice. This assay relies on supplying a microenvironment in which individual, antigen-specific lymphocytes are detected by antibody production. Although originally applied to determine the frequency of antigen-specific B cells within a milieu of non-limiting T-cell help, this assay has also been used to assess the precursor frequency of T helper cells which drive production of antibodies to nominal antigens (7,17). Accordingly, we reasoned that this assay might be adapted to quantitate the T cells capable of enabling autoreactive B cell activation and antibody formation. As shown in Fig. 1, lethally-irradiated recipient mice are reconstituted with non-limiting doses of syngeneic splenic B lymphocytes and increasing numbers of syngeneic CD4+ T lymphocytes. Eighteen hours later, recipient spleens are removed, chopped into 1 mm3 fragments, and these ‘fragments’ are individually cultured in vitro. Culture supernatants are collected 9 days later and assayed for anti-chromatin antibody production by ELISA (Fig. 1A).



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Fig. 1. Splenic fragment cultures. (A) Methods: recipient mice were lethally-irradiated and reconstituted with large numbers of syngeneic B cells and limiting numbers of syngeneic CD4+ T cells. Eighteen hours later, spleens were removed, chopped into 1 mm3 fragments, and each fragment individually cultured (92% O2, 8% C02) in one well of a 96-well plate for 9–12 days. (B) Anti-chromatin antibody production in splenic fragments requires CD4+ T cells. Sero-positive MRL/lpr recipient mice were lethally-irradiated. 5 x 107 MRL/lpr B cells and the indicated number of MRL/lpr T cells were transferred IV; 18 h later splenic fragments were produced. The percentage of wells positive by anti-chromatin ELISA is indicated. (Representative of two individual experiments.)

 
To validate application of the splenic focus assay to anti-chromatin antibody production, cultures were first generated with T and B cells from diseased, seropositive 6-month-old MRL-lpr mice. Even using seropositive mice, the induction of anti-chromatin antibodies by syngeneic B lymphocytes in the splenic focus assay required CD4+ T cells; no anti-chromatin antibody was detected in the culture supernatants when either B lymphocytes only or CD4+ T lymphocytes only were transferred (Fig. 1B). Thus, anti-chromatin antibody production is persistently T and B cell dependent and this technique can be used as a model system of autoantibody production from lupus-prone mice.

Pre-diseased, lupus-prone BWF1 mice have an elevated frequency of ANA-inducing CD4+ T lymphocytes
BWF1 mice develop a lupus-like syndrome with high titers of ANAs at 3–4 months of age (18). To obtain appropriate timepoints for our experiments, we first assayed sera from BWF1 mice for anti-chromatin antibodies. We chose non-autoimmune B6 mice as controls because parental NZB and NZW mice both develop autoimmunity (18). Sera from 6-month-old BWF1 mice contained high titers of anti-chromatin antibodies compared to similarly aged B6 mice (Fig. 2A). However, sera from young, 6-week-old BWF1 mice were seronegative and had no greater autoantibody titer than non-autoimmune B6 mice. Because they are not yet producing anti-chromatin antibodies, lymphocytes from young BWF1 mice were used to determine whether elevated autoreactive T-cell frequencies precede the onset of frank disease.



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Fig. 2. Pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T lymphocytes. (A) Young BWF1 mice are anti-chromatin negative. Serum from young (6 week) and old (6 month) BWF1 mice and young (6 week) B6 mice was assayed for the presence of IgG anti-chromatin antibodies by ELISA. The scatter plots show IgG anti-chromatin antibody responses of individual mice plotted against O.D. (B) The percentage of spleen fragments producing IgG anti-chromatin antibody is plotted against the number of CD4+ T lymphocytes transferred from young BWF1 or B6 mice. Representative results from one of four experiments. We calculate that the precursor frequency of autoantibody-inducing BWF1 CD4+ T lymphocytes is 1 per 105 CD4+ T cells (from 7 x 104 to 2 x 105). (C) Non-autoimmune B6 B cells can produce anti-chromatin antibodies. The percentage of B6 splenic fragments producing IgG anti-chromatin antibody is plotted against the number of CD4+ T lymphocytes transferred from young bm12 mice. Representative results from one of three experiments.

 
Splenic fragment limiting dilution cultures were initiated with non-limiting numbers of BWF1 B cells and increasing numbers of BWF1 CD4+ T cells from young BWF1 mice (see Fig. 1 for methodology). Parallel cultures were established using B6 CD4+ T and B cells. IgG was detected in all fragments containing non-limiting numbers of B cells and did not require CD4+ T cells (data not shown). In Fig. 2(B), the proportion of fragments that also produced anti-chromatin antibody is plotted against the number of CD4+ T cells transferred. Anti-chromatin antibody synthesis from BWF1 lymphocytes was dependent on the transfer of both CD4+ T (Fig. 2B) and B cells (data not shown). The number of responding BWF1 fragments increased when between 3.25 x 106 and 12.5 x 106 CD4+ cells were transferred. At greater doses of T cells, the percentage of responding fragments decreases, suggesting either an exhaustion of cellular resources or suppression of the autoantibody-inducing potential (19). In contrast, similar cultures set up with B6 CD4+ T cells minimally responded when at least 25 x 106 CD4+ T cells were transferred. Given the minimal response seen in B6 fragments, it was formally possible that both CD4+ T cells and B cells were limiting in these conditions. This would disagree with previous observations that the precursor frequency of autoreactive B cells is similar in autoimmune and non-autoimmune strains of mice (20). To formally exclude this possibility, B6 mice were reconstituted with non-limiting numbers of B cells and increasing numbers of alloreactive bm12 CD4+ T cells—this recapitulates the chronic graft-versus-host-disease model of murine lupus (21). As shown in Fig. 2(C), the number of responding B6 fragments increased with increasing numbers of alloreactive bm12 CD4+ T cells. Thus, BWF1 CD4+ T cells can drive autoantibody production, whereas B6 CD4+ T cells cannot.

The proportion of responding BWF1 spleen fragments is linearly related to the number of donor CD4+ T cells transferred, indicating that a single cell type is limiting (CD4+ T cells in this case, not B cells). This indicates that the responses within a fragment are initiated by a single T-cell clone, allowing determination of the frequency of anti-chromatin-inducing CD4+ T cells (7,13,17). To calculate the frequency of these active cells in the adoptively-transferred populations, we first determined the number of CD4+ T cells in each fragment culture by multiplying the homing efficiency of CD4+ T cells (see Methods) by the total number of fragment cultures analyzed. This number was then multiplied by the percentage of responding fragments at each cell dose to determine the precursor frequency of autoantibody-inducing CD4+ T cells. We calculate that in pre-disease BWF1 mice, 1 in 105 CD4+ T lymphocytes are capable of enabling antibody production by chromatin-specific B cells. In contrast, B6 CD4+ T cells were largely unable to induce anti-chromatin antibodies and the calculated precursor frequency is lower than 1 in 107. Thus, young, seronegative BWF1 mice have a >100-fold higher frequency of autoantibody-inducing CD4+ T lymphocytes than do B6 mice.

In vitro anti-chromatin responses are CD40L-dependent
Contact-dependent, MHC-restricted T–B interactions are required for the production of autoantibodies (2123), and disruption of CD40L–CD40 interactions by treatment with anti-CD40L attenuates autoantibody production in murine lupus (23). In order to assess whether the autoantibody production in splenic fragment assays reflected analogous cognate T–B interactions, we examined the dependence on CD40–CD40L interaction. Increasing numbers of BWF1 CD4+ T cells were transferred with non-limiting numbers of BWF1 B cells in the presence of anti-CD40L or control hamster IgG. The anti-chromatin antibody responses induced by BWF1 CD4+ T cells were completely blocked with anti-CD40L (Fig. 3). Thus, in vitro autoantibody production is contact-dependent and the splenic focus technique recapitulates in vivo results.



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Fig. 3. In vitro anti-chromatin antibody responses require CD40L. The percentage of spleen fragments producing IgG anti-chromatin antibody is plotted against the number of CD4+ T lymphocytes transferred from 6-week-old BWF1 mice in the presence of anti-CD40L ({alpha}CD40L) or control hamster IgG (hIgG). Representative results from three experiments.

 
Autoantibody-inducing CD4+ T-cell responses develop in the thymus
Recently, the argument that defective central tolerance contributes to the development of organ-specific autoimmunity has regained favor (46,24,25). It is not known if a similar deficit contributes to the development of systemic autoimmunity. To understand the origin of autoantibody-inducing CD4+ T cells in murine lupus, we asked if these CD4+ T cells are present in the thymus. Accordingly, CD4 SP thymocytes from BWF1 and B6 mice were isolated and assayed in splenic fragments. In Fig. 4, we show that BWF1 CD4 SP thymocytes can induce B cell production of anti-chromatin antibodies in a dose-dependent manner. Testing two doses of SP thymocytes does not allow calculation of a precursor frequency. However, SP thymocytes were much less efficient than peripheral T cells at inducing autoantibody production. More importantly, B6 CD4 SP thymocytes are largely unable to do so. Thus, defective thymic tolerance may contribute to the CD4+ T-cell repertoire enriched for autoantibody-inducing cells in BWF1 mice.



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Fig. 4. Autoantibody-inducing CD4+ T lymphocyte responses develop in the thymus. The percentage of spleen fragments producing IgG anti-chromatin antibody is plotted versus the number of CD4 SP thymocytes transferred from 6-week-old BWF1 or B6 mice. Results compiled from two separate experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Defective central tolerance contributes to systemic autoimmunity
We have found an increased frequency of autoantibody-inducing CD4+ T cells in the thymus and periphery of pre-diseased lupus-prone mice and argue that this predisposes BWF1 mice to the development of ANAs and systemic autoimmunity. This T cell-driven model of lupus is consistent with previous work suggesting that the limiting factor to autoantibody production is the availability of T-cell help. First, all murine models of lupus require CD4+ T cells (1416). Second, the chronic-graft-versus-host disease model of systemic autoimmunity is entirely driven by the activation of allogeneic CD4+ T cells in normally non-autoimmune mice (26). Third, the genetic susceptibility to lupus in multiple mouse models clearly includes intrinsic T-cell defects (27,28). Importantly, T-cell defects are also found in human lupus (29). However, many of these studies have not cleanly established whether altered CD4+ T-cell function precedes B cell dysfunction or arises secondarily. In contrast, the current investigation suggests that a CD4+ T-cell defect is primary in that it arises in the thymus.

Other groups have shown that central deletion of autoreactive thymocytes occurs normally in lupus-prone mice (3033). The differences could reflect three points. First, the previous studies examined only a few specificities of T cells (either T-cell receptor transgenic or superantigen-specific T cells) and the autoantibody-inducing CD4+ T cells might be different. Second, the splenic focus assay measures bona fide autoreactive T-cell responses in lupus (autoantibody production), not just deletion of double-positive thymocytes in response to high antigen dose, an often criticized approach (34). Third, the previous studies might not be suited to measure a subtle phenotype. In fact, normal T-cell selection is sensitive to subtle alterations of ligand density and T-cell antigen receptor signal intensity, and disruption of these processes results in autoimmunity (35,36). We believe that subtle alterations in thymic selection enriches the T-cell repertoire of lupus-prone mice with autoantibody-inducing T-cell specificities.

It is interesting that the frequency of autoantibody-inducing CD4+ T cells is higher in peripheral lymph node CD4+ T cells than in thymic CD4 SP cells (compare Figs 2B and 4). We cannot rule out the possibility that peripheral CD4+ T cells are simply more efficient ‘helper’ cells than SP thymocytes, which include a mixture of functionally immature and mature T cells. However, this difference probably reflects the peripheral expansion of autoantibody-inducing CD4+ T cells in lupus-prone BWF1 mice. In agreement, Anderson et al. (4) have argued that self-antigen-driven peripheral expansion of autoreactive CD4+ T cells enriches the peripheral repertoire of mice susceptible to organ-specific autoimmunity with self-reactive specificities. This result also suggests a defect in peripheral tolerance of autoantibody-inducing CD4+ T cells exists in BWF1 mice. Indeed, the failure of antigen-specific peripheral tolerance of CD4+ T cells in lupus-prone MRL-lpr mice has recently been described (37). Taken together, these studies suggest that a breach in central tolerance in association with peripheral expansion/maintenance of autoreactive CD4+ T cells contributes to both organ-specific and systemic autoimmunity.

Potential applications of the splenic focus assay
High-throughput biochemical screening can identify potent inhibitory compounds against a given target. The in vitro splenic focus technique could enable the high-throughput screening of therapeutics with utility in the treatment of lupus. Current lupus drug testing involves hundreds of intact mice and month- to year-long studies. The technique we describe allows drug testing on hundreds of individual fragment cultures from a few mice and a shorter time frame. We verified in a few weeks that anti-CD40L treatment blocked autoantibody production in hundreds of fragment cultures. Therefore, the splenic focus technique should be applicable to high-throughput biochemical screens for drug development in the treatment of lupus.

In addition, the splenic focus assay permits an analysis of the precursor frequency of autoantibody-inducing T (or autoantibody-producing B) lymphocytes in the naive repertoire of mice. Further, the ability to differentially isolate T lymphocytes, B lymphocytes and radioresistant elements allows a rapid dissection of the cell types responsible for a relevant lupus-associated phenotype. Lastly, antagonists and antibodies can be added to splenic fragment cultures to ascertain their applicability in blocking CD4+ T cell-induced autoantibody responses. Therefore, the splenic focus assay permits a potentially rapid dissection of the mechanisms of T–B cooperation during systemic autoimmune responses.

Conclusions
In this study, we show that young, pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T lymphocytes in their periphery. These CD4+ T cells are also present in the thymus, suggesting defective central tolerance of autoreactive CD4+ T lymphocytes. We believe that this increased frequency of autoantibody-inducing CD4+ T cells significantly contributes to BWF1 autoantibody production. We acknowledge, however, that one component of the genetic susceptibility of lupus is a B cell repertoire with lowered activation thresholds (38,39). It is likely that a T-cell repertoire enriched for autoantibody-inducing T lymphocytes and a B cell repertoire similarly tuned, synergize in the formation of ANAs and systemic autoimmunity. Future application of the splenic focus assay will permit a dissociation of the cell types responsible for the pathologic process.


    Acknowledgements
 
The authors thank Dr P. Cohen for helpful discussions, Dr A. Davidson (Albert Einstein College of Medicine, Bronx, New York) for providing aged BWF1 sera, and Dr L. Turka (University of Pennsylvania, Philadelphia, PA) for providing us with purified anti-CD40L (MR1). This work was supported by National Institutes of Health Grant AI48117 (to T.M.L.).


    Abbreviations
 
ANA—anti-nuclear antibody

BWF1—(NZB x NZW)F1

MRL/MpJ—FaslprMRL-lpr

SP—single-positive


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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