Anti-CD79α and anti-CD79β mAbs, but not other anti-B cell Abs, deplete MRL/lpr B cells
We screened several anti-B cell Abs in B6, B6/lpr, and MRL/lpr mice for in vivo B cell depletion by measuring splenic B220+sIgM+ B cells 1 wk after an injection of 1 mg anti-B cell Ab. Some anti-mouse B cell mAbs (anti-CD22 or anti-CD23) were entirely nondepleting even in B6 mice, whereas others (anti-CD19 and anti-CD45R/B220) depleted only in B6 and B6/lpr but not in MRL/lpr mice (). Anti-CD19 and anti-CD21 given alone (0.5 mg each, three weekly injections) or together did not affect MRL/lpr splenic B cell numbers (not shown). Daunomycin-conjugated Abs (anti-CD45R/B220, anti-CD23, or anti-CD24) injected at 0.3 mg per mouse weekly for 3 wk also failed to deplete B cells in MRL/lpr mice ( and data not shown). Despite the fact that some of these Abs were ineffective at depleting B cells, all of the mAbs could be demonstrated by FACS to have coated B cells in vivo (data not shown). Only anti-CD79β (HM79-16) was found to deplete splenic B cells in B6 and B6/lpr mice and in MRL/lpr mice 8 days after an injection of 1 mg Ab (). Additionally, B6 mice treated with increasing doses of anti-CD79β (0.1, 0.3, 0.5, or 1 mg) showed increasing amounts of B cell depletion (~40% depletion for 0.1 mg and 60% for 1 mg), with maximum depletion level achieved by 0.5 mg anti-CD79β (data not shown).
Decrease of splenic B cell numbers in anti-B cell Ab-treated micea
Subsequently, we also tested anti-CD79α (F11-172) and found that one injection of 0.5 mg anti-CD79α Ab reduced peripheral blood B cells in MRL/lpr mice to a similar extent as did 0.5 mg anti-CD79β alone or both Abs used in combination (data not shown), despite that these two Abs partially blocked each other in FACS staining (data not shown) and might compete for binding to B cells.
Thus, MRL/lpr mice were resistant to B cell-specific mAb-me-diated B cell depletion with the exception of anti-CD79α and anti-CD79β Abs.
Peripheral blood B cell depletion and recovery in MRL/lpr after a single dose of anti-CD79 Abs
To determine the best time for re-treatment, we measured peripheral blood B cell counts in MRL/lpr-IgHb mice following one injection of the combined anti-CD79 Abs (0.5 mg each of anti-CD79α and anti-CD79β), first every day (days 1–3) and then at 3-day intervals, for up to 24 days postinjection. Peripheral blood B cells were ~35% before treatment, declined starting day 1 postinjection, and reached a minimum of 10% on day 6 (), representing a maximum depletion of 75% B cells. B cell levels were then unchanged between days 6 and 9, but started to increase from day 9 and returned to pretreatment levels by 13 days postinjection (). Thus, weekly injections of these Abs might be required to maintain substantial B cell depletion in MRL/lpr mice.
FIGURE 1 B cell depletion and recovery in peripheral blood. MRL/lpr-IgHb mice were given one i.p. injection of 0.5 mg each of anti-CD79α and Anti-CD79β (AB) or PBS (n = 2 mice/group). Peripheral blood lymphocytes were collected and analyzed at (more ...)
To determine whether B cells that remained in circulation were fully coated by anti-CD79 Abs in vivo, we collected and stained cells with either FITC-anti-hamster Ab (hIgG), which revealed in vivo-acquired anti-CD79 Abs, or with FITC-anti-CD79β (CD79β), which revealed available free CD79 Ag on the cell surface. Taken together, these analyses would indicate whether resistance of B cells to depletion was due to insufficient surface coating of anti-CD79 Abs.
We found that on day 9 postinjection, the number of circulating B cells (B220+CD19 + ) were the same as those with surface hamster Ab (B220+hIgG+), but very few cells at this time point picked up ex vivo anti-CD79β Ab (). Thus, B cells found in circulation on day 9 postinjection were still fully coated with anti-CD79 Abs, suggesting that their lack of clearance at this point was not due to insufficient surface coating by the injected Abs.
After B cells had returned to preinjection levels (day 20), however, the circulating B cell profile was different from those on day 9. On day 20, few circulating B cells carried in vivo-bound anti-CD79 (B220+hIgG+), which was expected with the injected Ab concentration decreasing over time. Consistent with this, most remaining B cells bound ex vivo FITC-anti-CD79β Ab. However, their fluorescent intensity of ex vivo bound anti-CD79β was lower than cells from PBS-treated mice (). A similar pattern was seen with peripheral blood B cells collected on day 24 postinjection (), suggesting that after anti-CD79 treatment, CD79β low expression B cells were the first subset of cells to reenter the circulation.
Repeated anti-CD79 treatment depletes B cells from peripheral blood, spleen, lymph node, and bone marrow
To determine whether anti-CD79 Abs can maintain B cell depletion, MRL/lpr-Thy1.1 mice were treated with weekly injections of 0.5 mg each of anti-CD79α and anti-CD79β Abs for 17 wk (, Expt. 1). Control groups received 1 mg control hamster IgG or PBS. Peripheral blood B cells (B220+CD19+) remained ~75% depleted throughout the 17 wk of treatment (). Additionally, all remaining B cells showed in vivo-acquired anti-CD79α/β Abs (B220+hIgG+) but no binding to ex vivo anti-CD79β (B220+CD79b+) (). Serum anti-CD79β Ab levels were ~10 µg/ml just before each weekly injection (data not shown). Similar persistent B cell depletion and complete saturation of peripheral blood B cells by in vivo anti-CD79 Abs were found in additional studies where anti-CD79α and anti-CD79β Abs were administered weekly for a total of 9 or 11 wk (, Expt. II and Expt. III; data not shown).
FIGURE 2 Effects of weekly injection of anti-CD79 mAb on peripheral blood B cells. MRL/lpr-Thy1.1 mice were given weekly i.p. injections of either PBS, control hamster IgG Abs (Hamster IgG), or 0.5 mg each of anti-CD79α and Anti-CD79β (AB) for (more ...)
B cell depletion from lymphoid organs was analyzed in MRL/lpr-IgHb mice (, Expt. II) after 11 weekly treatments of anti-CD79 Abs. Anti-CD79-treated MRL/lpr-IgHb mice had 25% lower spleen weight as well as fewer lymphocyte () and >50% reduction of splenic B220+CD19+ B cells, B220+sIgM+ mature B cells, and B220+CD24+ immature B cells (). The extent of saturation by in vivo anti-CD79 Abs varied in anti-CD79-treated mice, with either complete or partial saturation by in vivo anti-CD79 (, AB-1 vs AB-2). Overall, the remaining B cells in spleen of anti-CD79-treated mice were saturated by in vivo anti-CD79 Abs, as B220+hIgG+ cells were similar in number to B220+CD19+ cells, but significantly greater than cells that bound anti-CD79β ex vivo Ab (B220+CD79b+) ().
FIGURE 3 B cell depletion from spleen. MRL/lpr-IgHb mice were given 11 weekly injections of PBS (n = 7) or 0.5 mg each of anti-CD79α and anti-CD79β (AB, n = 8) (, Expt. II) and were analyzed on week 12 for B cell depletion from lymphoid (more ...)
Lymph nodes of mice after 11 weekly anti-CD79 treatments showed >50% lower weight, total lymphocyte counts, ~60% reduction of B220+CD19+ B cells, B220+sIgM+ mature B cells, and a significant reduction of B220+CD24+ immature B cells (). Unlike splenic B cells, the remaining B cells in lymph nodes of anti-CD79-treated mice not only carried surface-bound hamster Ab (B220+hIgG+), but also picked up ex vivo anti-CD79β (). Indeed, the numbers of lymph node B220+hIgG+ and B220+CD79b+ cells were similar (), indicating that the resistant lymph node B cells were only partially saturated by in vivo anti-CD79 Abs.
FIGURE 4 B cell depletion from lymph nodes. Data from the same experiment as described in , Means and SEM of weight, lymphocyte numbers, and B cell numbers of lymph nodes. D, Representative anti-hamster IgG staining and binding to ex vivo Anti-CD79β (more ...)
Bone marrow of MRL/lpr-IgHb mice after 11 weekly anti-CD79 treatments also showed significant reduction in B220+CD19+ B cells, B220+sIgM+ mature B cells, and a significant reduction of B220+CD24+ immature B cells (). Similar to lymph node B cells and unlike splenic B cells, the remaining B cells in bone marrow were characterized by partial saturation by in vivo anti-CD79 Abs ().
FIGURE 5 B cell depletion from bone marrow. Data from the same experiment as described in , Percentages of bone marrow B cells. Shown are means and SEM. B, Representative anti-hamster IgG staining and binding to ex vivo Anti-CD79β by bone marrow (more ...)
In a separate study, we analyzed susceptibility of follicular and marginal zone B cell subsets to anti-CD79-mediated depletion in MRL/lpr-Thy1.1 mice given six weekly injections of anti-CD79 Abs (, Expt. IV). Similar to the longer treatment, total splenic B220+CD19+ B cells decreased ~60% in absolute numbers (), suggesting that there was no further depletion with longer treatment. Both follicular (FO) B cell numbers (CD19+CD23highCD21int) and marginal zone (MZ) B cells (CD19+CD23−CD21high) were depleted to a similar extent (FO, 78 ± 10% depletion; MZ, 90 ± 4% depletion) (). CD19+IgD+ B cells were also significantly depleted (). With most of the FO and MZ cells removed from spleen by anti-CD79, most (75%) of the remaining splenic CD19+ B cells were CD21low and CD23low newly formed (NF) B cells (NF, CD19+CD23lowCD21low) (, upper panel). However, there was no true expansion of the NF B cell subset, as their absolute numbers in spleens of anti-CD79-treated mice were the same as those in controls (, lower panel). Thus, NF B cells in MRL/lpr mice were apparently resistant to depletion by anti-CD79. We found that such resistance correlates with a lower expression level of CD79 molecules. In MRL/lpr controls, staining with anti-CD79β Ab along with anti-CD19, CD21, and CD23 revealed that NF B cell subsets showed lower fluorescence intensity of anti-CD79β binding than did FO and MZ B cells (). Consistent with this pattern of CD79β expression, NF B cells from anti-CD79-treated mice had less surface-bound hamster Ab (anti-CD79α and anti-CD79β) than did FO and MZ B cells (data not shown).
FIGURE 6 B cell subset sensitivity to anti-CD79-mediated depletion correlates with CD79β expression pattern. MRL/lpr-Thy1.1 female were given six weekly i.p. injections of either PBS or 0.5 mg each of anti-CD79α and Anti-CD79β Abs (AB) (more ...)
Changes in T cell subsets in MRL/lpr mice
Since congenitally B cell-deficient MRL/lpr mice showed decreased T cell-mediated inflammation in the kidney and decreased T cell activation, we asked whether anti-CD79 treatment is associated with reduced T cell activation. Thus, we enumerated T cell subsets from the spleen of MRL/lpr-Thy1.1 mice after 6 wk of anti-CD79 treatment (, Expt. IV). We found no significant change in splenic CD4 or CD8 T cell numbers but an unexpected expansion of double-negative T cells in both frequency and absolute number (), which had been reported to have regulatory functions.
FIGURE 7 Splenic T cell changes in anti-CD79-treated MRL/lpr. Data were from same experiment as described in . A, Relative frequency of T cell subsets. B, Absolute number of T cell subsets. C, Gating of activated, memory, and naive subsets based on CD62L (more ...)
Activated, memory, and naive subsets of CD4+ and CD8+ T cells were analyzed based on their CD62L and CD44 expression (). Naive (CD62LhighCD44low) CD4+ T cells in anti-CD79-treated mice rose significantly in relative frequency and expanded 2.5-fold in absolute cell number (). Most CD4 T cells in spleen of MRL/lpr mice were of memory phenotype (CD62LlowCD44high) and were unaffected by anti-CD79 treatment, as the absolute numbers of memory CD4+ T cells were unchanged in anti-CD79-treated mice (data not shown). The apparent decrease in percentage of memory CD4+ T cells in spleen of anti-CD79-treated mice was a result of the true expansion of naive CD4+ T cell (). Activated CD4+ T cells (CD62LhighCD44high) were the second largest CD4 T cell subset in the spleen of MRL/lpr mice and also did not show change by anti-CD79 treatment in either relative frequency () or absolute number (data not shown). Splenic CD8+ T cells showed no changes in the frequency of activated, memory, and naive T cell subsets (data not shown).
Thus, anti-CD79 treatment in MRL/lpr mice appeared to alter T cell phenotypes, particularly affecting CD4+ naive subsets and double-negative T cells without changing the frequency or absolute number of activated or memory CD4+ T cells.
Repeated anti-CD79 treatment reduced anti-chromatin IgG, prolonged survival, and reduced tissue inflammation
mice have a shortened lifespan due to systemic autoimmunity, with manifestations including high serum autoantibody levels, hyperimmunoglobulinemia, and inflammation in various organs (17
MRL/lpr mice develop high titers of autoantibodies, such as anti-chromatin and anti-dsDNA IgG. We found in three separate studies that the serum levels of anti-chromatin IgG were either suppressed or decreased over time by anti-CD79 treatment, whereas they rose steadily in control-treated mice (). However, serum levels of anti-dsDNA IgG were unaffected by anti-CD79 treatment (data not shown). Total serum IgG was also unaffected, although all mice exhibited hyperimmunoglobulinemia at the initiation of treatment (data not shown). Total serum IgM was also unaffected (data not shown).
FIGURE 8 Anti-CD79 treatment reduced anti-chromatin IgG. Serum anti-chromatin IgG level of MRL/lpr treated for 17 wk (A), 11 wk (B), or 9 wk (C) with PBS, 0.5 mg each of anti-CD79α and Anti-CD79β Abs (AB), or control Abs (Hamster IgG) ( (more ...)
Mouse anti-hamster IgG (MAHA) was significantly lower in anti-CD79α/β hamster IgG-treated MRL/lpr mice than in control hamster IgG-treated mice (data not shown). This finding suggests a suppressive effect of anti-CD79 Abs on the production of mouse Ab against foreign proteins.
Anti-CD79 treatment also improved survival rate and reduced the incidence of skin lesion development. The survival rates at week 17 for anti-CD79-treated mice (, Expt. I), control hamster IgG, and PBS groups were 83%, 20%, and 25%, respectively (p < 0.05 by χ2 test starting at week 14) (). All mice were free of skin lesions at the time of initiation of treatment. By week 11, 100% of the control mice had developed skin lesions, but only 38% of the anti-CD79-treated mice had skin lesions () (p < 0.05 by Fisher’s exact test, for week 11 and week 14). Since mortality was higher in control groups and may affect the evaluation of the incidence of skin lesions, we compared skin lesion development in survivors only and found significantly lower occurrence of skin lesions in the anti-CD79-treated group after 8 wk of treatment (). However, mice that developed skin lesions on anti-CD79 treatment did not become lesion-free with more treatment. Similar protective effects of anti-CD79 were observed in MRL/lpr-IgHb mice given 11 weekly anti-CD79 Abs (, Expt. II) (data not shown).
FIGURE 9 Survival rate and skin lesions. MRL/lpr-Thy1.1 mice were given weekly i.p. injections of PBS, control hamster IgG Abs (Hamster IgG), or 0.5 mg each of anti-CD79α and Anti-CD79β (AB) for 17 wk (, Expt. I). A, Survival rate. p < (more ...)
Improvement of survival might be due to not only to a systemic reduction of anti-chromatin autoantibodies and reduction of B cells, but also to the reduction in kidney injuries. The effect of anti-CD79 treatment on the kidney was evaluated in mice that underwent short-term (6-wk) treatment (, Expt. IV), during which time there was no significant mortality in the control groups. Kidneys of anti-CD79-treated mice had strikingly smaller () and less focal inflammatory infiltrates compared with the PBS control group (). Furthermore, anti-CD79 treatment significantly reduced interstitial nephritis, as fewer mice on anti-CD79 than in the control group scored >2 (, p = 0.0152, Fisher’s exact test). Glomerulonephritis was less severe in two of five (40%) mice in the anti-CD79 group, while all mice in PBS groups had scores >2 (). However, the difference was not significant.
FIGURE 10 Anti-CD79 reduced inflammation in kidney and submandibular salivary glands. MRL/lpr-Thy1.1 female were given six weekly injections of either PBS or anti-CD79α and anti-CD79β Abs (AB) (, Expt. IV; n = 5 mice/group). Shown are representative (more ...)
Salivary glands of MRL/lpr mice are known to develop extensive focal inflammatory infiltrates around blood vessels and ducts. MRL/lpr females treated for 6 wk with anti-CD79 Abs showed substantially smaller lesions and significantly lower density of inflammatory foci in their submandibular salivary glands ().