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Background Anti-carbohydrate antibody responses, including those of anti-blood group ABO antibodies, are yet to be thoroughly studied in humans. Because anti-ABO antibody-mediated rejection is a key hurdle in ABO-incompatible transplantation, it is important to understand the cellular mechanism of anti-ABO responses. We aimed to identify the main human B cell subsets that produce anti-ABO antibodies by analyzing the correlation between B cell subsets and anti-ABO antibody titers. Methods Blood group A-binding B cells were analyzed in peritoneal fluid and peripheral blood samples from 43 patients undergoing peritoneal dialysis and 18 healthy volunteers with blood group B or O. The correlation between each blood group A-specific B cell subset and anti-A antibody titer was then analyzed using Pearson's correlation analysis. Results Blood group A-binding B cells were enriched in CD27+CD43+CD1c- B1, CD5+ B1, CD11b+ B1, and CD27+CD43+CD1c+ marginal zone-B1 cells in peripheral blood. Blood group A-specific B1 cells (P=0.029 and R=0.356 for IgM; P=0.049 and R=0.325 for IgG) and marginal zone-B1 cells (P=0.011 and R=0.410 for IgM) were positively correlated with anti-A antibody titer. Further analysis of peritoneal B cells confirmed B1 cell enrichment in the peritoneal cavity but showed no difference in blood group A-specific B1 cell enrichment between the peritoneal cavity and peripheral blood. Conclusions Human B1 cells are the key blood group A-specific B cells that have a moderate correlation with anti-A antibody titer and therefore constitute a potential therapeutic target for successful ABO-incompatible transplantation.

Background Macrophages play important roles in xenograft rejection. Here, we investigated whether overexpression of human CD200 or CD47 in porcine endothelial cells (PEC) can suppress macrophages activation in xenogeneic immune responses. Methods PECs and human macrophages were incubated together, harvested, and analyzed for in vitro macrophage phagocytic and cytotoxicity activity, and cytokine release. Next, PECs were injected into renal subcapsular space of humanized mice. On day 10 posttransplantation, we analyzed xenograft survival and perigraft inflammatory cell infiltrations in PEC-to-humanized mouse transplantation. Results PECs highly expressing human CD200, CD47, or both CD47/CD200 were established by lentiviral vector transduction. Both CD200 and CD47 suppressed in vitro macrophage phagocytic and cytotoxic activity against PECs; decreased TNF-α, IL-1β, and IL-6 secretion; and increased IL-10 secretion. However, simultaneous overexpression of CD200 and CD47 did not show additive effects. Next, PECs were transplanted into NOD-scid IL-2Rg null mice, and human monocytes and lymphocytes were adoptively transferred 1 day after xenotransplantation. PEC xenograft cell death and apoptosis were decreased in the CD200-PEC and CD47/CD200-PEC groups. Perigraft infiltration of human T cells was suppressed by CD47; CD200 suppressed infiltration of human macrophages to a greater extent than CD47; and the CD47/CD200-PEC group exhibited the lowest level of leukocyte infiltration. In summary, overexpression of CD200 in PECs suppressed xenogeneic activation of human macrophages and improved survival of PEC xenografts in humanized mice; however, coexpression of CD200 and CD47 did not show additive effects. Conclusions Therefore, overexpression of human CD200 in donor pigs could constitute a promising strategy for overcoming xenograft rejection.

Plasmapheresis in combination with immunoglobulin and rituximab is often used to induce accommodation in ABO-incompatible (ABOi) living-donor transplantation; however, this regimen cannot be applied to cases of ABOi deceased-donor transplantation. Here, we investigated whether an anti-complement component 5 (C5) antibody-based regimen can induce accommodation in ABOi heart transplantation.Both IgM and IgG anti-blood type A antibodies were induced in wild-type mice by sensitization using human blood type A antigen. Heterotopic ABOi heart transplantation was performed from human blood type A-transgenic C57BL/6J mice to sensitized wild-type DBA/2 mice.Either anti-C5 antibody or conventional triple immunosuppressants (corticosteroid, tacrolimus, mycophenolate mofetil) alone did not induce accommodation in majority of ABOi heart allografts, whereas their combination induced accommodation in more than 70% of cases despite the presence of anti-A antibodies. The combination therapy markedly suppressed the infiltration of T cells and macrophages into ABOi allografts, despite mild deposition of IgG and C4d. T-cell activation and differentiation into Th1, Th2, and Th17 cells were suppressed along with CD49dCD4 T and follicular helper T cells in the combination treatment group. CD24 B cells, including both CD24CD23 marginal zone B cells and CD24CD23 T2-marginal zone B cells, were increased in the accommodation group.C5 inhibitor-based immunosuppression induced accommodation in murine ABOi heart transplantation, presenting a promising strategy for ABOi deceased-donor transplantation.

Adoptive transfer of regulatory T cells (Tregs) can delay disease progression and reduce mortality in lupus-prone mice. Here, we tested whether complex (IL-2C) consisting of IL-2 and anti-IL-2 monoclonal antibody (JES6-1) ameliorates lupus nephritis by expanding Tregs as an alternative to problematic Treg infusion therapy. IL-2C treatment of NZB/W F1 mice induced an effective and sustained expansion of CD4+CD25+Foxp3+ Tregs in both the kidneys and spleen along with decreased renal infiltration of T cells, B cells, and innate immune cells. Compared with controls, mice treated with IL-2C showed reduced proteinuria and fewer acute and chronic renal pathological lesions with improved renal function and survival. IL-2C significantly attenuated glomerular and tubular injury, vasculitis scores, and renal deposition of IgG and C3. Disease activity markers, such as high levels of anti-dsDNA antibodies and immunoglobulin levels, and low levels of complement were improved in sera of IL-2C-treated mice. IL-2C treatment decreased renal expression of TNF-α and IL-6, and the frequencies of IFN-γ+CD4+ and IL-17A+CD4+ T cells in both the kidneys and spleen. Depletion of Tregs by anti-CD25 antibodies abrogated the beneficial effects of IL-2C. When compared with combination therapy of steroid and mycophenolate mofetil, IL-2C treatment showed similar or better outcomes. Thus, IL-2C protected lupus-prone mice against lupus nephritis by expanding Tregs. Hence, IL-2C could have therapeutic potential in lupus nephritis.

BACKGROUND: Regulatory B cells are a newly discovered B cell subset that suppresses immune responses. Recent studies found that both anti-CD45RB and anti–Tim-1 treatments regulate immune responses by inducing regulatory B cells; however, the role of these cells in renal ischemia-reperfusion injury (IRI) is unknown. METHODS: Using mouse models, including T cell–deficient (RAG1 knockout and TCRα knockout) mice and B cell–deficient (μMT) mice, we investigated the effects of regulatory B cells and anti-CD45RB on IRI and the mechanisms underlying these effects. RESULTS: Adoptive transfer of regulatory B cells before or after IRI attenuated renal IRI. Anti-CD45RB treatment with or without anti–Tim-1 before IRI increased renal infiltration of CD19(+)Tim-1(+) regulatory B and regulatory T cells. Anti-CD45RB decreased serum creatinine levels, pathologic injury score, tubular apoptosis, and proinflammatory cytokines levels, whereas IL-10 levels increased. Following IRI, anti-CD45RB with or without anti–Tim-1 also induced regulatory B cells, improving renal function and tubular regeneration. In RAG1 knockout mice with B cell transfer, TCRα knockout mice, and wild-type mice with T cell depletion, anti-CD45RB increased regulatory B cells and attenuated IRI. However, anti-CD45RB did not attenuate IRI in RAG1 knockout mice with T cell transfer or μMT mice and induced only mild improvement in wild-type mice with B cell depletion. Furthermore, B cell–deficient mice receiving B cells from IL-10 knockout mice (but not from wild-type mice) did not show renal protection against IRI when treated with anti-CD45RB. CONCLUSIONS: Anti-CD45RB treatment attenuated acute renal injury and facilitated renal recovery after IRI through induction of IL-10(+) regulatory B cells, pointing to anti-CD45RB as a potential therapeutic strategy in renal IRI.

The major obstacle to successful ABO blood group-incompatible kidney transplantation (ABOi KT) is antibody-mediated rejection (AMR). This study aimed to investigate transcriptional profiles through RNA sequencing and develop a minimally invasive diagnostic tool for discrimination between accommodation and early acute AMR in ABOi KT. Twenty-eight ABOi KT patients were selected: 18 with accommodation and 10 with acute AMR at the 10th day posttransplant protocol biopsy. Complete transcriptomes of their peripheral blood were analyzed by RNA sequencing. Candidate genes were selected by bioinformatics analysis, validated with quantitative polymerase chain reaction, and used to develop a classification model to diagnose accommodation. A total of 1385 genes were differentially expressed in accommodation compared with in AMR with P-adjusted < .05. Functional annotation and gene set enrichment analysis identified several immune-related and immunometabolic pathways. A 5-gene classification model including COX7A2L, CD69, CD14, CFD, and FOXJ3 was developed by logistic regression analysis. The model was further validated with an independent cohort and discriminated between accommodation and AMR with 92.7% sensitivity, 85.7% specificity, and 91.7% accuracy. Our study suggests that a classification model based on peripheral blood transcriptomics may allow minimally invasive diagnosis of acute AMR vs accommodation and subsequent patient-tailored immunosuppression in ABOi KT.

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase-1 (HO-1) or soluble tumor necrosis factor-α receptor type I with human IgG1 Fc (sTNF-αR-Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO-1, sTNF-αR-Fc, sTNF-αR-Fc/HO-1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood-derived CD34(+) stem cells into NOD-scid-IL-2Rγ(null) mice. Both HO-1 and sTNF-αR-Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig-to-humanized mice transplantation. The sTNF-αR-Fc/HO-1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor-α and IL-6 in treatment groups; however, frequency of pig-specific interferon-γ-producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO-1 or sTNF-αR-Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO-1 or sTNF-αR-Fc transgenic pigs have potential for islet xenotransplantation.

The Th cells that regulate peritoneal B-1 cell functions have not yet been well characterized. To address this question, we investigated peritoneal CD4+ T cells, observed a high frequency of the conjugates of B-CD4+ T cells in the peritoneal cavity, and identified a population of CD49dhighCD4+ T cells that constituted about half of all CD4+ T cells in the peritoneal cavity, but were rarely found in other compartments. Peritoneal CD49dhighCD4+ T cells were CD44highCD62Llow; expressed integrin α4β1 and CXCR3; and rapidly secreted IFN-γ, TNF-α, and IL-2, showing features of proinflammatory Th1 cells. Peritoneal CD49dhighCD4+ T cells developed spontaneously, were detected at the age of 12 d, and showed stem cell–like properties. Their development was observed in mice deficient for signaling lymphocytic activation molecule-associated protein, but not in athymic nude mice and mice lacking in expression of MHC class II on thymic epithelial cells. Peritoneal CD49dhighCD4+ T cells were more resistant to irradiation and more sensitive to NAD-induced cell death than CD49dlowCD4+ T cells. Notably, peritoneal CD49dhighCD4+ T cells also showed some characteristics of follicular Th cells, such as the expression of programmed cell death 1, ICOS, IL-21, and CXCR5. Moreover, peritoneal CD49dhighCD4+ T cells enhanced the secretion of IgM Abs by B-1a cells and IgG Abs by splenic B cells. These data suggest that peritoneal CD49dhighCD4+ T cells may be innate-like CD4+ T cells, which develop early and have a dual capacity to support both humoral and cellular immunity.

Introduction B cell subtypes and immunoglobulin variable (V), diversity (D), joining (J) gene segment usage of B cell receptors in ABO-incompatible (ABOi) kidney transplantation (KT) in comparison to ABO-compatible KT have not been studied. The aims of this study were to analyze the VDJ gene segment usages of B cell receptors in peripheral blood of ABOi KT recipients. Methods Eighteen ABOi KT patients with accommodation (ABOiA), 10 ABO-compatible stable KT patients (ABOcS), and 10 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10 after transplantation were selected. Complete transcriptomes of their peripheral blood samples were sequenced and analyzed through RNA sequencing. Results By family, immunoglobulin heavy chain variable 3 (IGHV3), immunoglobulin light kappa chain variable 1 (IGKV1), immunoglobulin light lambda chain variable 2 (IGLV2), and immunoglobulin light lambda chain joining 3 (IGLJ3) gene segments were most frequently used in all groups, and their usage was not statistically different among the three groups except for IGHV3 and IGKV1. IGKV1 was more frequently used in the ABOiA group than in the ABOcS group. According to individual gene segments, IGHV3-7, IGHV3-15, IGHV4-59, IGKV3-11, IGLV1-44, IGLV2-14, IGLV4-69, and IGLV7-46 were more frequently used in the ABOcS group than other groups, and IGKV3-7 was more frequently used in the ABOiR group than other groups. IGLV5-52 and IGLV7-43 were more frequently used in the ABOiA group than in ABOcS group. Conclusions Our findings suggest that RNA sequencing transcriptomic analyses of peripheral blood can provide information on the VDJ gene usage of B cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT.

Background Granulocyte colony-stimulating factor (G-CSF) can induce regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs). Despite the immune modulatory effects of G-CSF, results of G-CSF treatment in systemic lupus erythematosus are still controversial. We therefore investigated whether G-CSF can ameliorate lupus nephritis and studied the underlying mechanisms. Methods NZB/W F1 female mice were treated with G-CSF or phosphate-buffered saline for 5 consecutive days every week from 24 weeks of age, and were analyzed at 36 weeks of age. Results G-CSF treatment decreased proteinuria and serum anti-dsDNA, increased serum complement component 3 (C3), and attenuated renal tissue injury including deposition of IgG and C3. G-CSF treatment also decreased serum levels of BUN and creatinine, and ultimately decreased mortality of NZB/W F1 mice. G-CSF treatment induced expansion of CD4+CD25+Foxp3+ Tregs, with decreased renal infiltration of T cells, B cells, inflammatory granulocytes and monocytes in both kidneys and spleen. G-CSF treatment also decreased expression levels of MCP-1, IL-6, IL-2, and IL-10 in renal tissues as well as serum levels of MCP-1, IL-6, TNF-α, IL-10, and IL-17. When Tregs were depleted by PC61 treatment, G-CSF-mediated protective effects on lupus nephritis were abrogated. Conclusions G-CSF treatment ameliorated lupus nephritis through the preferential expansion of CD4+CD25+Foxp3+ Tregs. Therefore, G-CSF has a therapeutic potential for lupus nephritis. Electronic supplementary material The online version of this article (doi:10.1186/s12882-016-0380-x) contains supplementary material, which is available to authorized users.

Extracellular adenosine triphosphate (ATP) binds to purinergic receptors and, as a danger molecule, promotes inflammatory responses. Here we tested whether periodate-oxidized ATP (oATP), a P2X7 receptor (P2X7R) antagonist can attenuate renal ischemia-reperfusion injury and clarify the related cellular mechanisms. Treatment with oATP prior to ischemia-reperfusion injury decreased blood urea nitrogen, serum creatinine, the tubular injury score, and tubular epithelial cell apoptosis after injury. The infiltration of dendritic cells, neutrophils, macrophages, CD69 + CD4 + , and CD44 + CD4 + T cells was attenuated, but renal Foxp3 + CD4 + Treg infiltration was increased by oATP. The levels of IL-6 and CCL2 were reduced in the oATP group. Additionally, oATP treatment following injury improved renal function, decreased the infiltration of innate and adaptive effector cells, and increased the renal infiltration of Foxp3 + CD4 + Tregs. Post-ischemia-reperfusion injury oATP treatment increased tubular cell proliferation and reduced renal fibrosis. oATP treatment attenuated renal functional deterioration after ischemia-reperfusion injury in RAG-1 knockout mice; however, Treg depletion using PC61 abrogated the beneficial effects of oATP in wild-type mice. Furthermore, oATP treatment after transfer of Tregs from wild-type mice improved the beneficial effects of Tregs on ischemia-reperfusion injury, but treatment after transfer of Tregs from P2X7R knockout mice did not. Renal ischemia-reperfusion injury was also attenuated in P2X7R knockout mice. Experiments using bone marrow chimeras established that P2X7R expression on hematopoietic cells rather than non-hematopoietic cells, such as tubular epithelial cells, plays a major role in ischemia-reperfusion injury. Thus, oATP attenuated acute renal damage and facilitated renal recovery in ischemia-reperfusion injury by expansion of Tregs.

Background Studies on B-cell subtypes and V(D)J gene usage of B-cell receptors in kidney transplants are scarce. This study aimed to investigate V(D)J gene segment usage in ABO-incompatible (ABOi) kidney transplant (KT) patients compared to that in ABO-compatible (ABOc) KT patients. Methods We selected 16 ABOi KT patients with accommodation (ABOiA), 6 ABOc stable KT patients (ABOcS), and 6 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10, whose graft tissue samples had been stored in the biorepository between 2010 and 2014. Complete transcriptomes of graft tissues were sequenced and analyzed through RNA sequencing (RNA-seq). The international ImMunoGeneTics information system (IMGT®) was used for in-depth comparison of V(D)J gene segment usage. Results The mean age of the 28 KT recipients was 43.3 ± 12.8 years, and 53.6% were male. By family, IGHV3, IGHJ4, IGLV2, and IGLJ3 gene segments were most frequently used in all groups, and their usage was not statistically different among the three patient groups. While IGKV3 was most frequently used in both the ABOiA and ABOiR groups, IGKV1 was most commonly used in the ABOcS group. In addition, while IGKJ1 was most commonly used in the ABOiA and ABOcS groups, IGKJ4 was most frequently used in the ABOiR group. According to individual gene segments, IGHV4–34 and IGHV4–30-2 were more commonly used in the ABOiR group than in the ABOiA group, and IGHV6–1 was more commonly used in the ABOcS group than in the ABOiR group. IGLV7–43 was more commonly used in the ABOcS group than in the ABOi group. However, technical variability, small sample size, and potential confounding effects of Rituximab or HLA mismatching are limitations of our study. Conclusions Our findings suggest that RNA-seq transcriptomic analyses can provide information on the V(D)J gene usage of B-cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT.

Background: Current therapies for advanced renal cell carcinoma (RCC) have low cure rates or significant side effects. It has been reported that complexes composed of interleukin (IL)-2 and stimulating anti-IL-2 antibody (IL-2C) suppress malignant melanoma growth. We investigated whether it could have similar effects on RCC. Methods: A syngeneic RCC model was established by subcutaneously injecting RENCA cells into BALB/c mice, which were administered IL-2C or phosphate-buffered saline every other day for 4 weeks. RCC size was measured serially, and its weight was assessed 4 weeks after RENCA injection. Immune cell infiltration into RCC lesions and spleen was assessed by flow cytometry and immunohistochemistry. Results: IL-2C treatment increased the numbers of CD8(+) memory T and natural killer (NK) cells in healthy BALB/c mice (P < 0.01). In the spleen of RCC mice, IL-2C treatment also increased the number of CD8(+) memory T, NK cells, and macrophages as compared to PBS-treated controls (P < 0.01). The number of interferon-gamma-and IL-10-producing splenocytes increased and decreased, respectively after 4 weeks in the IL-2C-treated mice (P < 0.01). Tumor-infiltrating immune cells including CD4(+) T, CD8(+) T, NK cells as well as macrophages were increased in IL-2C-treated mice than controls (P < 0.05). Pulmonary edema, the most serious side effect of IL-2 therapy, was not exacerbated by IL-2C treatment. However, IL-2C had insignificant inhibitory effect on RCC growth (P = 0.1756). Conclusions: IL-2C enhanced immune response without significant side effects; however, this activity was not sufficient to inhibit RCC growth in a syngeneic, murine model.

Adoptive transfer of regulatory T cells (Tregs) can delay disease progression and reduce mortality in lupus-prone mice. Here, we tested whether complex (IL-2C) consisting of IL-2 and anti-IL-2 monoclonal antibody (JES6-1) ameliorates lupus nephritis by expanding Tregs as an alternative to problematic Treg infusion therapy. IL-2C treatment of NZB/W F1 mice induced an effective and sustained expansion of CD4

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.

CD138, known as a marker of plasma cells, was reported to be expressed to an intermediate level in the murine bone marrow precursor B cells. Here an intermediate level of CD138 expression was also noted in a subpopulation of splenic follicular B cells, which were distinguishable from CD138(high) plasma cells, whereas the majority of transitional or marginal zone B cells did not express CD138. These CD138(int) B cells were IgM(low)IgD(high) mature B cells, located within follicular B cell zone, and expressed a lower level of CD21 than CD138(-) follicular B cells. During in vitro culture of splenic cells, the proportion of CD138(int) B cells increased, which was noticeably reversed by the addition of IL-4 to the culture. The experiments with sorted CD138(int) cells showed that IL-4-mediated regulation of the CD138 expression was B cell-intrinsic and independent of in vitro B cell death. Our results demonstrate that mouse CD138(int) B cells characterize a subpopulation of IgM(low)IgD(high) mature follicular B cells. The CD138 expression on follicular B cells may represent a reversible status, reflecting a dynamic state probably influenced by IL-4.

B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5(+) B-1a and CD5(-) B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.