Coordinated Circulating T Follicular Helper and Activated B Cell Responses Underlie the Onset of Antibody-Mediated Rejection in Kidney Transplantation.

BACKGROUND: Although antibody-mediated rejection (ABMR) has been long recognized as a leading cause of allograft failure after kidney transplantation, the cellular and molecular processes underlying the induction of deleterious donor-specific antibody (DSA) responses remain poorly understood. METHODS: Using high-dimensional flow cytometry, in vitro assays, and RNA sequencing, we concomitantly investigated the role of T follicular helper (TFH) cells and B cells during ABMR in 105 kidney transplant recipients. RESULTS: There were 54 patients without DSAs; of those with DSAs, ABMR emerged in 20 patients, but not in 31 patients. We identified proliferating populations of circulating TFH cells and activated B cells emerging in blood of patients undergoing ABMR. Although these circulating TFH cells comprised heterogeneous phenotypes, they were dominated by activated (ICOS+PD-1+) and early memory precursor (CCR7+CD127+) subsets, and were enriched for the transcription factors IRF4 and c-Maf. These circulating TFH cells produced large amounts of IL-21 upon stimulation with donor antigen and induced B cells to differentiate into antibody-secreting cells that produced DSAs. Combined analysis of the matched circulating TFH cell and activated B cell RNA-sequencing profiles identified highly coordinated transcriptional programs in circulating TFH cells and B cells among patients with ABMR, which markedly differed from those of patients who did not develop DSAs or ABMR. The timing of expansion of the distinctive circulating TFH cells and activated B cells paralleled emergence of DSAs in blood, and their magnitude was predictive of IgG3 DSA generation, more severe allograft injury, and higher rate of allograft loss. CONCLUSIONS: Patients undergoing ABMR may benefit from monitoring and therapeutic targeting of TFH cell-B cell interactions.

Disparate Recruitment and Retention of Plasmacytoid Dendritic Cells to The Small Intestinal Mucosa between Young and Aged Mice.

Plasmacytoid dendritic cells (pDC), a highly specialized class of innate immune cells that serve as rapid sensors of danger signals in circulation or in lymphoid tissue are well studied. However, there remains knowledge gaps about age-dependent changes of pDC function in the intestinal mucosa. Here, we report that under homeostatic conditions, the proportion of pDC expressing C-C chemokine receptor 9 (CCR9) in the intestinal intraepithelial cell (iIEC) population is comparable between young (2-4 months) and aged (18-24 months) mice, but the absolute numbers of iIEC and pDC are significantly lower in aged mice. Employing the classic model of acute endotoxemia induced by lipopolysaccharide (LPS), we found a decrease in the proportion and absolute number of intraepithelial pDC in both young and aged mice despite the LPS-induced increased expression of the chemokine C-C ligand 25 (CCL25), the ligand of CCR9, in the intestinal mucosa of young mice. In adoptive transfer experiments, a significantly lower number of pDC was retained into the intestinal layer of aged host mice after LPS administration. This was associated with recoverable pDC numbers in the intestinal lumen. Furthermore, co-adoptive transfer of young and aged pDC into young hosts also showed significantly lower retention of aged pDC in the epithelial layer compared to the co-transferred young pDC. Collectively, these data show age-associated changes in mucosal CCL25 gene expression and in pDC number. These may underlie the reported inadequate responses to gastrointestinal pathogens during chronologic aging.

Pregnancy-induced humoral sensitization overrides T cell tolerance to fetus-matched allografts in mice.

Immunological tolerance to semiallogeneic fetuses is necessary to achieving successful first pregnancy and permitting subsequent pregnancies with the same father. Paradoxically, pregnancy is an important cause of sensitization, resulting in the accelerated rejection of offspring-matched allografts. The underlying basis for divergent outcomes following reencounter of the same alloantigens on transplanted organs versus fetuses in postpartum females is incompletely understood. Using a mouse model that allows concurrent tracking of endogenous fetus-specific T and B cell responses in a single recipient, we show that semiallogeneic pregnancies simultaneously induce fetus-specific T cell tolerance and humoral sensitization. Pregnancy-induced antibodies, but not B cells, impeded transplantation tolerance elicited by costimulation blockade to offspring-matched cardiac grafts. Remarkably, in B cell-deficient mice, allogeneic pregnancy enabled the spontaneous acceptance of fetus-matched allografts. The presence of pregnancy-sensitized B cells that cannot secrete antibodies at the time of heart transplantation was sufficient to precipitate rejection and override pregnancy-established T cell tolerance. Thus, while induction of memory B cells and alloantibodies by pregnancies establishes formidable barriers to transplant success for multigravid women, our observations raise the possibility that humoral desensitization will not only improve transplantation outcomes, but also reveal an unexpected propensity of multiparous recipients to achieve tolerance to offspring-matched allografts.

T-bet+CD27+CD21- B cells poised for plasma cell differentiation during antibody-mediated rejection of kidney transplants.

Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high-dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21- activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR- patients and at baseline levels in DSA- patients. RNA-Seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of interferon regulatory factor 4 and Blimp1, and upon coculture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21-dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.

Impact of Induction Therapy on Circulating T Follicular Helper Cells and Subsequent Donor-Specific Antibody Formation After Kidney Transplant.

INTRODUCTION: The cellular events that contribute to generation of donor-specific anti-HLA antibodies (DSA) post-kidney transplantation (KTx) are not well understood. Characterization of such mechanisms could allow tailoring of immunosuppression to benefit sensitized patients. METHODS: We prospectively monitored circulating T follicular helper (cTFH) cells in KTx recipients who received T-cell depleting (thymoglobulin, n = 54) or T-cell nondepleting (basiliximab, n = 20) induction therapy from pre-KTx to 1 year post-KTx and assessed their phenotypic changes due to induction and DSA occurrence, in addition to healthy controls (n = 13), for a total of 307 blood samples. RESULTS: Before KTx, patients displayed comparable levels of resting, central memory cTFH cells with similar polarization to those of healthy controls. Unlike basiliximab induction, thymoglobulin induction significantly depleted cTFH cells, triggered lymphopenia-induced proliferation that skewed cTFH cells toward increased Th1 polarization, effector memory, and elevated programmed cell death protein 1 (PD-1)int/hi expression, resembling activated phenotypes. Regardless of induction, patients who developed DSA post-KTx, harbored pre-KTx donor-reactive memory interleukin (IL)-21+ cTFH cells and showed higher % cTFH and lower % of T regulatory (TREG) cells post-KTx resulting in elevated cTFH:TREG ratio at DSA occurrence. CONCLUSION: Induction therapy distinctly shapes cTFH cell phenotype post-KTx. Monitoring cTFH cells before and after KTx may help detect those patients prone to DSA generation post-KTx.

Phenotype, function, and differentiation potential of human monocyte subsets.

Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

B Cells, Antibodies, and More.

B cells play a central role in the immunopathogenesis of glomerulonephritides and transplant rejection. B cells secrete antibodies that contribute to tissue injury via multiple mechanisms. In addition, B cells contribute to disease pathogenesis in autoimmunity and alloimmunity by presenting antigens as well as providing costimulation and cytokines to T cells. B cells also play an immunomodulatory role in regulating the immune response by secreting cytokines that inhibit disease onset and/or progression. B cell-targeted approaches for treating immune diseases of the kidney and other organs have gained significant momentum. However, much remains to be understood about B-cell biology in order to determine the timing, duration, and context of optimal therapeutic response to B cell-targeted approaches. In this review, we discuss the multifaceted roles of B cells as enhancers and regulators of immunity with relevance to kidney disease and transplantation.

B cells mediate chronic allograft rejection independently of antibody production.

Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells.

Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient µMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology.

BACKGROUND: Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts. METHODS: We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG(-/-)) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection. RESULTS: Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG(-/-) mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection. CONCLUSIONS: The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.

Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation.

BACKGROUND: Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes. METHODS: We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied. RESULTS: We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells. CONCLUSIONS: Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

The polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response.

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4(+) T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4(+) T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice.

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.

Role of secondary lymphoid tissues in primary and memory T-cell responses to a transplanted organ.

Secondary lymphoid tissues are the hub of adaptive immune responses wherein rare cognate lymphocytes encounter dendritic cells bearing antigen from peripheral tissues and differentiate into effector and memory cells that eliminate antigen. It is accepted that immune responses against microbial and tumor antigens are initiated within secondary lymphoid tissues. There is less agreement on whether the same principle applies to immune responses to a transplanted organ because an allograft expresses foreign major histocompatibility complex and contains donor antigen presenting cells that could activate T cells directly in situ leading to rejection. Recent studies confirm that although naïve T cells can be primed within the allograft, their differentiation to effect rejection is dependent on secondary lymphoid tissues. Antigen-experienced memory T cells, unlike Naïve T cells, function largely independent of secondary lymphoid tissues to cause allograft rejection. In an alloimmune response, secondary lymphoid tissues support not only immune activation but also immune regulation essential for allograft survival. Here, we will review recent findings and discuss the role of secondary lymphoid tissues in primary and memory alloimmune responses.

The yin and yang of B cells in graft rejection and tolerance.

Various lineages of B cells are being increasingly recognized as important players in the etiology and prognosis of both acute and chronic graft rejection. The role of immature, chronically activated B cells, as efficient antigen-presenting cells, supporting recalcitrant cell-mediated graft rejection and late lineage B cells driving humoral rejections, is being increasingly recognized. This review captures the recent literature on this subject and discusses the various roles of the B cell in renal graft rejection and conversely, also in graft tolerance, both in animal and human studies. In addition, novel therapies targeting specific B-cell lineages in graft rejection are also discussed, with a view to developing more targeted therapies for graft rejection.

Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs.

Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.

CD70 signaling is critical for CD28-independent CD8+ T cell-mediated alloimmune responses in vivo.

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.

Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs.

The allospecifc T cell population responding to a transplanted organ consists of both naive and memory lymphocytes. Although it is established that naive T cells are activated by antigen within the organized structures of secondary lymphoid organs (the spleen, lymph nodes, and mucosal lymphoid tissues), it is not clear whether memory T cell activation and propagation depend on homing to these organs. To answer this question, we investigated whether allospecific naive or memory T cells can mediate acute cardiac allograft rejection in mutant mice that lack all of their secondary lymphoid tissues. The results of our experiments demonstrated that antigen-experienced memory T cells have two advantages over naive T cells: (i) memory T cells mount a vigorous immune response that leads to allograft rejection independent of secondary lymphoid organs; and (ii) memory T cells generate more memory T cells without homing to secondary lymphoid organs. These unique properties of memory T cells were further confirmed by showing that memory-like T cells that arise from the homeostatic proliferation of naive T cells in the absence of antigenic stimulation are suboptimal at rejecting allografts and do not generate memory T cells in mice devoid of secondary lymphoid tissues.