Effects of specific anti‐B and/or anti‐plasma cell immunotherapy on antibody production in baboons: depletion of CD20‐ and CD22‐positive B cells does not result in significantly decreased production of 
anti‐αGal antibody

Abstract

Anti-Galalpha1-3Gal antibodies (antialphaGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alphaGal oligosaccharide columns temporarily depletes antialphaGal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of antialphaGal Ab following EIA. The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 mAb (proportional to CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and antialphaGal Ab production were assessed both in vitro and in vivo in baboons (n = 9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LN), staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and antialphaGal Ab levels were measured daily. In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in antialphaGal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in antialphaGal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM, > 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in antialphaGal Ab after EIA. Proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum antialphaGal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline antialphaGal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in antialphaGal Ab production were obs...

Reconstitution of B cells after B cell–depletion therapy: Comment on the article by Leandro et al

“…Thus, although the majority of reconstituted B cells are naive B cells, our results suggest that the reconstitution of B cells in peripheral blood is derived from 2 different, simultaneously occurring pathways: 1) proliferation and differentiation of immature B cells from the bone marrow and 2) migration of memory B cells out of secondary lymphoid organs. The identification of these 2 mechanisms of B cell reconstitution is consistent with the findings from a study of baboons showing incomplete depletion of CD20ϩ B cells in the lymph nodes after rituximab treatment (4). Whether tissue depletion of B cells is complete after rituximab treatment in humans is unknown, but the lower expression of CD20 on B cells from bone marrow and lymph nodes versus peripheral blood indicates that further study is clearly needed (5 …”

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

“…The increased activation state of GC B cells during cGVHD has the potential to make monoclonal cellular depletion therapies less effective, as seen in other autoimmune disorders. 28,29 The production of class-switched antibodies requires the ability of T cells to localize to the GC and provide survival signals to B cells during cGVHD. Interruption of trafficking of T cells to the B-cell follicle by CXCR5 was sufficient for preventing GC response and preventing cGVHD (Figure 3).…”

Rituximab therapy and autoimmune disorders: Prospects for anti–B cell therapy

“…Moreover, the B cells involved in an autoimmune disease may also be less accessible to the effects of rituximab. In fact, data from nonhuman primates with cross-reactive CD20 indicate that treatment with certain anti-CD20 antibodies may be much less effective at depleting B cells in lymph nodes (37), but published data on this topic are currently limited. Presumably, these findings are also relevant to the effects at sites of tissue infiltration (e.g., joints), where disease-associated lymphocytes may, in fact, preferentially localize in different disorders (9).…”