Inflammation Causes Resistance to Anti-CD20-Mediated B Cell Depletion.

B cells play a central role in antibody-mediated rejection and certain autoimmune diseases. However, B cell-targeted therapy such as anti-CD20 B cell-depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through toll-like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B cell pool by bone marrow-derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity.

B cells help alloreactive T cells differentiate into memory T cells.

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B-cell-deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T-cell proliferation and activated CD8 T-cell survival. These results indicate that B cells help alloreactive T-cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.

Type I interferons are not critical for skin allograft rejection or the generation of donor-specific CD8+ memory T cells.

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8+ T-cell responses in mice that lack the IFN-I receptor (IFN-IR-/-). We found that IFN-IR-/- mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR-/- male skin was transplanted to syngeneic IFN-IR-/- female mice. Quantitation of the male (H-Y)-specific CD8+ T-cell response in these mice revealed normal generation of donor-specific CD8+ effector T cells but fourfold reduction in CD8+ memory T cells. Memory CD8+ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR-/- mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8+ T cells into long-lived, functional memory T cells.

The roles of CD8 central and effector memory T-cell subsets in allograft rejection.

The contribution of secondary lymphoid tissue-homing central memory T cells (T(CM)) and peripheral tissue-homing effector memory T cells (T(EM)) to allograft rejection is not known. We tested whether T(EM) is the principal subset responsible for allograft rejection due to the nonlymphoid location of target antigens. Skin allograft rejection was studied after transferring either CD8 T(CM) or T(EM) to wild-type mice and to mice that lack secondary lymphoid tissues. We found that CD8 T(CM) and T(EM) were equally effective at rejecting allografts in wild-type hosts. However, CD8 T(EM) were significantly better than T(CM) at rejecting allografts in the absence of secondary lymphoid tissues. CD8 T(CM) were dependent upon secondary lymphoid tissues more than T(EM) for optimal differentiation into effectors that migrate into the allograft. Recall of either CD8 T(CM) or T(EM) led to accumulation of T(EM) after allograft rejection. These findings indicate that either CD8 T(CM) or T(EM) mediate allograft rejection but T(EM) have an advantage over T(CM) in immune surveillance of peripheral tissues, including transplanted organs.

Tertiary lymphoid tissues generate effector and memory T cells that lead to allograft rejection.

Tertiary lymphoid tissues are lymph node-like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin-alpha(RIP-LT alpha), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP-LT alpha allografts that harbor tertiary lymphoid tissues are rejected, while wild-type allografts that lack tertiary lymphoid tissues are accepted. Wild-type allografts transplanted at the same time as RIP-LT alpha skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP-LT alpha skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T-cell activation.