Female CBA/J (cross between a Bagg albino female and a DBA male), C57BL/6 (cross between a female N.57 and a male N.52 from the A. Lathrop’lab) and MRL/lpr mice (composite genome derived 75.0% from LG/J, 12.6% AKR/J, 12.1% C3H/HeDi and 0.3% C57BL/6J) were purchased from Harlan-France or Charles River-France/Jackson Laboratory. Genotyping of mice (Fas gene) was done using the primers and PCR protocols provided by The Jackson laboratories for MRL/lpr mice. In most of experiments including those conducted to explore TCR and BCR V-J rearrangements, 11-13 week-old female MRL/lpr mice received a single intravenous (i.v.) administration of P140 peptide in saline or saline alone as control. MHC-matched CBA/J mice were used as healthy control. For testing the effect of P140 on the ability of MRL/lpr mice to mount an immune response to a foreign immunogen, 14 week-old female CBA/J and MRL/lpr mice received P140 at weeks 5, 7, 9, and 13 (100µg in 100µL saline per mouse) by the i.v. route, and then ovalbumin (OVA) (Sigma-Aldrich, ref. A5503) at weeks 14, 16 and 18 (100µg in 100µL saline, emulsified in complete Freund’s adjuvant (FA) for the first injection and in incomplete FA for the subsequent ones) by the subcutaneous route. They were bled at weeks 14 (control), 17 and 19. For continuous labeling experiments with bromodeoxyuridine (BrdU) in vivo, mice received first an intraperitoneal injection of BrdU (1mg/20g body weight; Sigma-Aldrich, ref. B5002) and then BrdU in drinking water for 10 days (0.8mg/mL). They received a single i.v. dose of P140 5 days after they received intraperitoneal injection of BrdU. Cell subsets were analyzed by flow cytometry.

Proteinuria, dermatitis and levels of antibodies to dsDNA were recorded in MRL/lpr mice (and CBA/J mice as control) as described (25). Urine protein levels were assessed semi-quantitatively using albumin reagent strips (0 = none, 1 = 30 to 100mg/dL, 2 = 100 to 300mg/dL, 3 = 300 to 1000mg/dL, 4 = >1000mg/dL; Albustix, Miles Scientific). The titer of circulating dsDNA antibodies was defined as the inverse of the dilution giving an absorbance value of 1.0 in our ELISA conditions (25).

Throughout this study, the mouse murine T cell receptor β-chain (mTRB) VJ and immunoglobulin heavy (H) chain VJ (IGH VJ) genes are designed according to the international ImMunoGeneTics database (http://www.imgt.org). Samples were analyzed with ImmunTraCkeR kits, using the ImmunID IFS platform (ImmunID). Detailed protocols and expression of results have been published previously (41, 42). Briefly, three days after peptide (100µg per mouse) or placebo treatment, genomic DNA was extracted from CBA/J and MRL/lpr PBMCs or spleen cells. For each sample, multi-N-plex PCR was performed using an up-stream primer specific of all functional members of a given TRBV family and a downstream primer specific of a given TRBJ segment. An independent multi-N-plex PCR was also applied for IGHV and IGHJ amplification. These assays allow for the simultaneous detection of several V–J rearrangements in the same reaction. Using this technique, it was possible to detect 209 different mTRBV–TRBJ and 92 mIGHV-IGHJ rearrangements. All V–J1, J2, and Jn PCR products were separated according to their size. PCR products were separated on a 0.8%-agarose gel, directly stained with SYBR Green I, and quantified using a charge-coupled device camera equipped with a BIO-1D quantification software (Vilbert Lourmart). Constel ID software (ImmunID) was used for further analytical studies including the generation of three-dimensional repertoire illustration. Results are expressed as percentages of detected rearrangements among the total 209 mTRB and 92 mIGH possible combinatorial rearrangements. Of note, due to high number of sub-members in the V1 family, the latter one has been splitted into 8 tubes (V1a to V1h). IGH V1a comprises sub-members: V1-11, 12, 15, 67; IGH V1b: V1-49, 63; IGH V1c: V1-17-1, 18, 26, 34, 22, 62-2, 71, 76; IGH V1d: V1-14, 47, 80; IGH V1e: V1-5, 7, 4; IGH V1f: V1-20, 31, 37, 39, 42, 43, 66, 75, 77, 84, 78; IGH V1g: V1-9, 19, 36, 62-1, 58; IGH V1h: V1-50, 52, 53, 54, 55, 56, 59, 61, 62-3, 64, 69, 72, 74, 81, 82, 85. For the other IGH Vn families, they comprise all corresponding members.

The following antibodies were used: for the BrdU experiments, fluorescein isothiocyanate (FITC)-labeled CD3 monoclonal antibody (mAb) clone 145-2C11, peridinin chlorophyll (PerCP)-cyanin 5.5-B220 mAb clone RA3-6B2, phycoerythrin (PE)-CD138 mAb clone 281.2; for defining the cell subtypes that repopulate the peripheral blood, PerCP-Cy5.5-CD3 mAb clone 145-2C11, allophycocyanin-B220 mAb clone RA3-6B2, FITC-Ly6G mAb clone 1A8, PE-Gr1 mAb clone RB6-8C5, allophycocyanin-CD138 mAb clone 281.2, PerCP-Cy5.5 -CD19 mAb clone 1D3; for the B cell studies, FITC-CD19 mAb clone 1D3 and allophycocyanin-CD138 mAb clone 282-2 (all from BD PharMingen). For evaluating lymphocyte viability, splenocytes were stained with PE-Cy7-CD3e (Invitrogen, ref. 25-0031-82), eFluor506-CD45R/B220 (Invitrogen, ref. 69-0452-82), Alexa Fluor (AF) 700-CD4 (Invitrogen, ref. 56-0041-82), allophycocyanin-Cy7-CD8a (Molecular Probes, ref. A15386), PerCP-Cy5-CD25 (Invitrogen, ref 45-0251-82) and FITC-FOXP3 (Invitrogen, ref. 11-4776-42) mAbs. For the comparison of leukocytes in WT and T-deficient MRL/lpr mice, staining was done with FITC-TCRαβ (Invitrogen, ref. 11-5961-82), PE-TCRγδ (Invitrogen, ref. 12-5711-82), PerCP-Cy5.5-Ly6C (Invitrogen, ref. 45-5932-82), Pe-Cy7-CD45 (Invitrogen, ref. 25-0451-82), allophycocyanin-CD8a (Invitrogen, ref. 17-0081-82), AF700-CD3 (Invitrogen, ref. 56-0032-82), allophycocyanin-Cy7-CD4 (Invitrogen, ref. A15384), eFluor506-Ly-6G/Ly-6C (Invitrogen, ref. 69-5931-82), SB600-CD25 (Invitrogen, ref. 63-0251-82), SB702-B220 (Invitrogen, ref. 67-0452-82), PerCP-Cy5.5-CD19 (Invitrogen 45-0193-82) mAbs, and LIVE/DEAD Fixable Violet stain Super Bright 436 (Invitrogen, ref. L34955). Stained cells were analyzed on an Attune NxT cytometer (Invitrogen) and FlowJo software (TreeStar).

Spleen cells of 11-13 week-old female MRL/lpr, CBA/J and C57BL/6 mice were cultured in 24-well plates (2x10⁶ cells/well) were incubated for 24h at 37°C with increasing concentrations of P140. They were stained and analyzed by flow cytometry as above.

Intracellular cell staining was performed using the allophycocyanin-labeled BrdU flow kit (BD, ref. 552598). Briefly, 0.5x10⁶ cells per condition were stained with surface markers, washed and permeabilized using cytofix/cytoperm solution and cytoperm permeabilization buffer plus. Cells were washed and incubated with DNase (30µg per 10⁶ cells). In a last condition, cells were stained with anti-BrdU-allophycocyanin-labeled antibody, 20min at room temperature, washed and analyzed by cytometry.

The method used was as described previously (18, 43). The number of leukocytes/mL in the peripheral blood was evaluated by counting cells five days after a single injection of P140 peptide (100µg/mouse) into MRL/lpr or CBA/J mice. White total cells and cell subsets were analyzed by flow cytometry.

For measuring IL-2, IL-4, IL-6, TNFα, IFNγ, IL-17a and IL-10 serum levels, we used the BD cytometric beads array mouse Th1, TH2, Th17 kit (BD, ref. 560485) according to the manufacturer protocol. Briefly, 25µL of MRL/lpr sera were diluted 1:1 with assay diluent, incubated with 50µL of capture beads and 50µL of Mouse Th1/Th2/Th17 PE detection reagent. Assay tubes were incubated for 2h at room temperature and protected from light. After one wash with 1 mL of wash buffer, beads were resuspended with 300µL of wash buffer and acquired on a FACSCalibur cytometer. A standard curve established with known concentrations of each cytokine was used for quantification.

Splenic B cells were isolated from MRL/lpr mice by negative selection (Pan B cell isolation kit mouse; Miltenyi Biotec, ref. 130-104-443). Biotinylated anti-CD138 antibodies (1µg/mL; clone 281-2, BD Pharmingen) were added to deprive preexisting PCs. MRL/lpr B cells (97-99% purity, as estimated by flow cytometry) were then let to differentiate for 5 days in the presence of anti-CD40 antibody (0.5µg/mL; BD Pharmingen, ref. 4045566), IL-21 (10ng/mL; R&D, ref. 594-ML-010) and increasing concentrations of P140. Antibodies secretion was measured by ELISpot as previously described with minor modifications (44). Briefly, 96-well multiscreen plats (Millipore) were coated 1h at 37°C with anti-mouse IgM or IgG (1µg/mL; Jackson ImmunoResearch) in phosphate-buffered saline (PBS), pH 7.4. After washing with PBS, membranes were saturated for 1h with RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 0.05mM β-mercaptoethanol, 10µg/mL gentamycin and 10 mM HEPES. Serial dilutions of differentiated B cell suspensions were incubated for 18-24h at 37°C. The last steps were as described (18) using anti-mouse IgM (µ chain-specific) biotin-labeled Ig or anti-mouse IgG (Fcγ-specific) biotin-labeled Ig (both 1:20,000 in PBS containing 0.05% (v/v) Tween (PBS-T); Jackson ImmunoResearch) that were added for 12h at 4°C, followed by alkaline phosphatase-labeled extravidin (Sigma-Aldrich) for 1h at 37°C and 5-bromo-4-chloro-indolyl phosphate and nitro-blue tetrazolium chloride substrate (Sigma-Aldrich). The reaction was stopped with water when spots were clearly visible. Spots were counted with a Bioreader4000 (BioSysGmbH). The results were expressed as the number of IgM or IgG-secreting cells/10⁶ total cells using the following formula: (number of spots/well – background measured in control wells incubated with PBS or BSA) x dilution factor of the cell suspension.

Animal protocols were carried out with the approval of the local Institutional Animal Care and Use Committee (CREMEAS, Strasbourg, France) and the French Ministère de l’Enseignement Supérieur de la recherche et de l’innovation (APAFiS#2016112916066575 and APAFiS#23210-2019120617587704). According to our agreement, and taking into account the best European practices in the field, we took the necessary measures to avoid pain and minimize the distress and useless suffering of mice during the time of experiment and killing process.

Statistical analyses were performed using Wilcoxon Rank Sum tests (comparison of diversity between two groups), nonparametric Mann-Whitney test, paired or unpaired t-tests when sample distribution followed a Gaussian distribution. P value <0.05 were considered statistically significant.

We previously showed that P140 administration into MRL/lpr mice causes egress of several immune cell subtypes from the peripheral blood of treated mice without affecting T cell priming and the ability of P140-teated mice to resist to an infectious viral challenge (18, 22, 23, 39). This effect was very specific and was not seen with the numerous P140 analogues that were tested (18). To analyze further the characteristics of remaining cells, we first studied this egress from a dynamic viewpoint. The depleting effect of P140 was peptide dose-dependent until a threshold dose of 100µg P140/mouse/injection, which led to a peripheral cell balance similar to the one measured in healthy mice ( Figure 1A ). Increasing P140 dose at 200µg P140/mouse did not affect this balance further. Using the optimal dose of 100µg/mouse, we found that at day 3 post-P140 treatment, there was a drop of T cell (including DN T cell), B cell, PC, monocyte and granulocyte counts ( Figures 1B–D ; Supplementary Figure 1 ). On day 10 post-P140 treatment, the counts of monocytes and granulocytes still remained low, in contrast to the counts of T cells, DN T cells, B cells and PCs that reached again the basal level measured in untreated MRL/lpr mice.

To investigate the functional status of T and B cells in the “depleted” compartment, then we measured the incorporation of BrdU in various immune cell subsets of CBA/J and MRL/lpr mice that received or not P140. Three groups of five mice each, were tested, namely MRL/lpr mice that received NaCl only (group 1), MRL/lpr mice that received P140 in saline (group 2) and control CBA/J mice that received NaCl only (group 3). Mice received a first i.v. injection of BrdU and then permanently, in drinking water, for 10 days. P140 was given once, intravenously, on day 5. On day 10, flow cytometry analysis of cell subtypes showed that in the spleen, although there were fewer T cells in group 2 compared to group 1, there was no change in the number of dividing T cells (BrdU^high) ( Figure 1E ). As expected the number of cycling cells was elevated in the MRL/lpr DN T cell subset with regard to normal mice but these numbers were not different in treated and non-treated animals ( Supplementary Figure 2A ). In the PBMC fraction, in contrast, the total number of non-dividing (long-lived) T cells was not different in the three groups ( Figure 1F ), while the number of BrdU^high T cells, which was raised in MRL/lpr mice compared to normal mice, was significantly reduced upon P140 treatment ( Figure 1F ). The proliferation of blood circulating DN T cells was apparently not modified upon P140 treatment ( Supplementary Figure 2B ).

Regarding the B cell compartment, BrdU pulse chase experiments showed that the number of dividing and non-dividing MRL/lpr B cells was decreased upon P140 treatment both in the spleen and PBMC fraction ( Figures 1G, H ). Although no change was found in the spleen, fewer dividing PBs and PCs were counted in the peripheral blood ( Supplementary Figures 2C– F ). It has to be noticed however that although these results are statistically significant, they should be approached with cautious since the number of PCs and PBs was very small.

Together, these analyses indicate that following a single i.v. administration of P140, the proliferative status of total MRL/lpr T cells evaluated 5 days later is globally not changed in the secondary lymphoid tissue (spleen), while it is affected in the peripheral blood compartment. In parallel, the number of proliferating B cells is reduced both in the spleen and peripheral blood compartments. The proliferating rate of PBs and PCs, which remains broadly stable upon P140 administration tended to diminish in the peripheral blood.

The above data suggest that post-P140 treatment (one single i.v. injection), there is a window of about 10 days during which immune cell abnormalities seem to be reduced in the peripheral blood of MRL/lpr mice. Over this period, we found that while the level of blood-circulating IL-2, IL-4, IL-6, IL-10 and IFN-γ showed no or marginal variations in treated vs. untreated mice ( Figure 2 ), there was a sharp significant drop of soluble IL-17a levels in P140-treated mice. Type-I interferon levels were not tested here since in contrast to other mouse models of lupus, MRL/lpr mice do not show evidence of type-I IFN signature (45).

These result, together with previous data showing that upon treatment with P140, a number of cytokine-encoding genes are significantly up- or down-regulated in non-activated lymph node CD4⁺ T cells (23), underline that the profile of secreted cytokines is shifted upon P140 treatment.

To gain relevant information regarding the characteristics of circulating lymphocytes that remain in the blood of mice shortly after P140 administration (“depleting” phase), next we analyzed the respective TCR and BCR repertoire of circulating T and B cells. The genomic DNA was prepared from PBMCs and spleen cells, 3 days post-P140 treatment, i.e. within the time window of P140 effect identified above at the periphery. Because of lack of documented information in the existing literature, the first step of this investigation was to compare TCR and BCR V-J rearrangements in CBA/J and MRL/lpr mice, which share the same MHC and mTRB haplotypes (46), and thereafter, the possible influence of P140 on these rearrangements. The experiments and the results are described in full detail in the supplementary material. The most relevant results are summarized below.

A thorough analysis of mTRB and mIGH diversity and combinatorial repertoire composition was performed from spleen and PBMC samples of CBA/J and MRL/lpr mice. We used a general immune companion diagnostic assay to monitor T and B cell responses and evaluate the immune status in the different strains of mice (41). The results were compared for each group of mice and for each repertoire. A total of 43 spleen and 44 PBMC samples were analyzed for mTRB repertoire, and 43 spleen and 39 PBMC samples were analyzed for mIGH repertoire ( Supplementary Table 1 ). When CBA/J and MRL/lpr mice samples were compared, no statistically significant difference was observed in terms of mTRB and mIGH combinatorial diversity ( Figure 3 ; Supplementary Figures 3A, B, D, E ). However, the analyses of VJ rearrangements showed the existence of some specific mTRB VJ rearrangements that were statistically more frequently represented in MRL/lpr mice compared to CBA/J mice and could therefore be designed as a signature. No notable mIGH VJ repertoire signature was revealed (For full details see Supplementary material; Supplementary Figures 4, 5 ; Supplementary Tables 1–3 ).

Next, we examined the influence of P140 on these rearrangements. When untreated and P140-treated MRL/lpr mice were compared, no difference was observed in splenocytes and PBMCs in terms of mTRB VJ combinatorial diversity ( Figures 3A, B ; Supplementary Figures 3B, C ). The analysis done on the entire mTRB VJ repertoire ( Supplementary Figures 4 ) revealed that compared to untreated MRL/lpr mice, one mTRB VJ rearrangement only in spleen and none in PBMCs was significantly more frequent in P140-treated mice ( Supplementary Figures 6A ; Supplementary Table 4 ). However, mTRB VJ rearrangements were found more frequently in untreated MRL/lpr mice compared to P140-treated mice. From those mTRB VJ rearrangements, V29-J2.1 and V3-J2.3, which were found both in splenocytes and PBMCs from untreated mice, could represent a signature that is absent in P140-treated mice. Regarding the fate of the mTRB VJ rearrangements distinguishing MRL/lpr spleen from CBA/J mice, some (V3-J2.3, V26-J2.4, V29-J2.1) also present a significant difference of frequency between treated and untreated MRL/lpr mice, meaning that they are apparently affected by the treatment. The analysis done with PBMC samples led to the same conclusions, which concern mTRB VJ rearrangements V29-J2.5 and V29-J1.5. These findings collectively suggest that pre- and post-P140 treatment, the distribution of frequencies of some mTRB VJ rearrangements is changed. To reinforce this assumption, we therefore compared the repertoire composition of the P140-treated MRL/lpr mice versus CBA/J control mice ( Supplementary Figure 7 ; Supplementary Table 5 ). While P140 seemed to have no influence on the representation of certain rearrangements (see the details in the supplementary material), the frequency of others (V3-J2.3, V26-J2.4, and V29-J2.1 in spleen and V29-J2.5 and V29-J1.5 in PBMCs) were found levels that were very similar in P140-treated mice and CBA/J mice (no significant difference of frequency could be highlighted for these rearrangements between the two groups; Supplementary Figure 7A ). These results highlight that treatment of MRL/lpr mice with P140 has an impact on the distribution frequency of rare mTRB VJ rearrangements observed in MRL/lpr prior to treatment, which reach levels similar to those found in healthy CBA/J mice.

With regard to mIGH VJ repertoires of MRL/lpr mice treated or not with P140 ( Supplementary Figures 4C, D ) the comparison revealed that more frequent differences occur in PBMCs than in spleens, where 22 rearrangements were found significantly more often in P140-treated than in untreated MRL/lpr mice ( Supplementary Figure 6B ; Supplementary Table 4B ). As also found in the case of mTRB VJ rearrangements, the distribution of the frequency of mIGH VJ rearrangements was affected by P140 treatment. However, since in our hands no mIGH VJ repertoire signature could be visualized in MRL/lpr B cells, it was not possible to investigate a potential effect of peptide on this specific repertoire with regard to the one of healthy mice (see details in Supplementary Figure 7B ; Supplementary Table 5B ).

To further decipher the downstream aftermath of P140, next we looked at whether P140 could modify the capacity of MRL/lpr B cells to differentiate into CD138/Syndecan-1⁺ PBs and PCs. We addressed this question in vitro using purified B cells from 12-week-old MRL/lpr mice, which were let to differentiate for 5 days in the presence of anti-CD40 antibody and IL-21 (47, 48). The number of CD19^-CD138⁺ PCs was too low in the cultures to support any reliable conclusion ( Figures 4A, B ). However, we observed that the percentage of CD19⁺CD138⁺ PBs diminished in a P140 concentration-dependent manner ( Figures 4C, D ). A scrambled control analogue (ScP140) had no measurable effect. These results corroborate our previous flow cytometry results (23) showing the effect of P140 on CD138⁺B220^- PCs, measured 20h after MRL/lpr PBMCs treatment, and data generated with cells from lupus patients treated ex vivo with P140 and ScP140 (40).

Since the size of CD19⁺CD138⁺ PBs compartment was significantly reduced in the presence of P140, we assessed the effect on Ig production and the number of antibody-secreting cells. The latter, which represent the terminally differentiated cells of the humoral immune response, are the main effector B cells that expand in acute SLE (49). Of note, autophagy has been seen to play a general role in the late stages of B cell activation and subsequent PC differentiation (50–52). In the experimental in vitro setting we applied, naïve B cells were not able to efficiently class-switch and therefore the fine specificity of secreted Ig could not be identified. However, ELISpot results clearly indicated that in a P140 concentration-dependent manner, the frequency of IgM-secreting cells was significantly diminished in the culture ( Figure 4E ). ScP140 had no measurable effect.

Considering the changes of immune cell distribution and phenotypes described above, at this stage it was important to ensure that P140-treated mice were still able to mount a classical response to a foreign antigen. We immunized untreated and P140-treated MRL/lpr mice with soluble OVA used as a model immunogen and evaluated the antibody response by ELISA ( Figure 5A ). A strong anti-OVA IgG antibody response was measured in all mice of each study group. Compared to healthy mice, the mean anti-OVA antibody titers were particularly elevated in MRL/lpr mice after the second administration of immunogen, and then tend to decrease ( Figure 5B ). This hyper-responsiveness was not seen in MRL/lpr that received P140 prior immunization. With the exception of this feature, the mean anti-OVA antibody titers were of the same order in immunized untreated and P140-treated MRL/lpr mice. The data corroborate our previous observations indicating that P140 treatment did not affect the capacity of MRL/lpr mice challenged with an infectious dose of influenza virus given intranasally, to mount specific T- and B-cell responses against viral antigens and to recover from infection (39). Taken together, these past and present data convincingly support that the immune cells and associated molecular mechanisms are competent following administration of P140 to mice and able to develop an appropriate immune response to a given antigen. This indicates that P140 does not behave as an immunosuppressant and instead would help restore immune homeostasis.

Previous data have shown that in vivo, P140 has no direct effect on post-BCR signaling of memory, naïve mature, transitional and B1 cells (40). Among other outcomes (22, 24, 40), these findings led us to propose that the effect of P140 likely occurs via a mechanism involving upstream steps of T cell regulation (T cell activation, proliferation and ability of signaling) with a strong downstream impact on B cell maturation and differentiation into antibody-secreting cells ( Figure 5C ). This scheme is further comforted here by our findings that in vitro, P140 has no direct impact on precursors (CD3⁺, B220^-, CD4⁺, CD8^-, CD25^- , FOXP3⁺) and mature (CD3⁺, B220^-, CD4⁺, CD8^-, CD25⁺, FOXP3⁺) Tregs from MRL/lpr and healthy mice of different haplotypes ( Supplementary Figure 8 ).

In this study, we confirm even further the sequence of events of the P140’ mechanism of action in showing that in the absence of activated peripheral CD4⁺ T cells, P140 exerts no protecting effect in MRL/lpr mice ( Figure 5D ; Supplementary Figures 9, 10 ). We unexpectedly identified a phenotypic drift of our in-house colony, which was a progeny of mice obtained from the Jackson Laboratory (MRL/MpJ-Faslpr/J mice; stocking number #00485). These mice displayed both cellular and clinical differences compared to wild-type (WT) MRL/lpr mice. While they retained expected genotype of WT MRL/lpr mice, as attested by classical genotyping of Fas gene by PCR ( Figure 5D ), and several pathophysiological characteristics of the WT MRL/lpr strain (splenomegaly, enlarged lymph nodes and salivary glands; Supplementary Figures 9A–C ), they strikingly present a milder disease with a reduced level of proteinuria ( Supplementary Figure 9D ) and a decreased count of peripheral cells compared to MRL/lpr mice ( Figure 5D ; Supplementary Figure 10 ). The levels of circulating IgG antibodies to ds DNA were elevated in both, WT and T-cell defective MRL/lpr mice (titers were ~100, 1680 and 2145 in CBA/J, WT and T-deficient MRL/lpr mice, respectively, as measured by ELISA and calculated at an absorbance value of 1.0 in ELISA). We noted that in contrast to WT MRL/lpr mice, which often have a few (1, 2) successive litters of pups only, T-cell defective MRL/lpr mice had greater number of births (a full description of these mice will be published elsewhere). Detailed study of peripheral immune cell subsets showed that the myeloid lineage (granulocytes and monocytes) remained similar in the WT and spontaneously-derived MRL/lpr mice. However, in the latter the lymphoid lineage cells were significantly affected in the peripheral blood ( Supplementary Figure 10 ). The number of circulating CD4⁺, CD8⁺, and DN T cells was significantly lower compared to WT MRL/lpr mice. Overall, the number of activated CD25⁺CD4⁺ T cells that are characteristic of the WT MRL/lpr mouse, was greatly reduced, and found at the baseline observed in healthy CBA/J mice ( Figure 5D ; Supplementary Figure 10 ). The compartment of unconventional T cells expressing a γδ TCR (a relatively small subset of T cells in peripheral blood that can be activated by lipids and phosphoantigens) was affected just like T cells expressing TCR α- and β-chains. An important result is that T-cell deficient MRL/lpr mice appeared totally refractory to P140 treatment ( Figure 5D ), which reinforces our operational scheme ( Figure 5C ). This finding is consistent with previous results generated in WT MRL/lpr mice depleted in γδ T cells (23). It corroborates past results showing the effects resulting from the depletion of cells of the lymphoid lineage on the course of the disease in WT MRL/lpr mice (38, 53, 54).