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BACKGROUND The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen—a 10% l-proline–stabilized IVIG product—that allows extensive reduction of isoagglutinin concentrations in the final product. STUDY DESIGN AND METHODS An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry–based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. RESULTS The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. CONCLUSION Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

BACKGROUND Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti-A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti-A/B isoagglutinin titers of the final IVIG product. STUDY DESIGN AND METHODS A donor screening method for anti-A isoagglutinins using an automated indirect agglutination test (IAT) in gel cards was developed. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen (human 10% liquid IVIG). Anti-A/B isoagglutinin levels were measured by IAT, direct agglutination test, and a flow cytometry–based assay. RESULTS Screening of plasma from 705 randomly selected donors identified 6.8% donors with high anti-A isoagglutinin titers in plasma. Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in laboratory-scale pooled plasma. The same donor screening method applied to industry-scale production resulted in exclusion of 5.1% of donors and produced a one-titer-step reduction of both anti-A and anti-B isoagglutinin titers in the final IVIG product. CONCLUSION Anti-A/B isoagglutinin titers in IVIG products can be reduced on an industrial scale through implementation of anti-A donor screening, which may lower the risk of hemolysis after IVIG therapy.

γδ T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vγ9Vδ2 + T cells (termed γδ T-APCs) to cross-present microbial and tumor antigens to CD8 + αβ T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that γδ T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8 + αβ T cells. Induction of APC functions in Vγ9Vδ2 + T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of γδ T cells irrespective of their state of activation. γδ T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4 + αβ T cell responses by both types of APCs. Our study reveals unexpected properties of human γδ T-APCs in the induction of CD8 + αβ T effector cells, and justifies their further exploration in immunotherapy research.

Human Vγ9/Vδ2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3+ cells. This “transitional response,” occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in αβ T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vγ9/Vδ2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic αβ T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate γδ T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of γδ T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vγ9/Vδ2 TCR agonists.

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product.An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures.The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process.Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti-A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti-A/B isoagglutinin titers of the final IVIG product.A donor screening method for anti-A isoagglutinins using an automated indirect agglutination test (IAT) in gel cards was developed. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen (human 10% liquid IVIG). Anti-A/B isoagglutinin levels were measured by IAT, direct agglutination test, and a flow cytometry-based assay.Screening of plasma from 705 randomly selected donors identified 6.8% donors with high anti-A isoagglutinin titers in plasma. Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in laboratory-scale pooled plasma. The same donor screening method applied to industry-scale production resulted in exclusion of 5.1% of donors and produced a one-titer-step reduction of both anti-A and anti-B isoagglutinin titers in the final IVIG product.Anti-A/B isoagglutinin titers in IVIG products can be reduced on an industrial scale through implementation of anti-A donor screening, which may lower the risk of hemolysis after IVIG therapy.

Human γδ T cells are considered to play a vital role in protective immunity through cytokine secretion and cytotoxic activity. We report that cells expressing the Vγ2Vδ2 + –T cell receptor (Vδ2 + T cells) also display principal characteristics of professional antigen-presenting cells such as dendritic cells. Thus, when activated, these cells efficiently processed and displayed antigens and provided co-stimulatory signals sufficient for strong induction of naïve αβ T cell proliferation and differentiation. We suggest that, upon microbial activation, Vδ2 + T cells participate in the induction of adaptive immune responses and that these cells may be a useful tool in vaccine development and immunotherapy.

Chemotactic migration of leucocytes largely depends on adhesive interaction with the substratum and recognition of a chemoattractant gradient. Both aspects, cell adhesion and chemotaxis, are regulated by members of the family of chemotactic cytokines (chemokines) comprising structurally related and secreted proteins of 67-127 amino acids in length. Breakdown in the control of leucocyte mobilisation contributes to chronic inflammatory diseases and, hence, interference with chemokine function is a promising approach for the development of novel anti-inflammatory medication. Chemokines target all types of leucocyte, including haematopoietic precursors, mature leucocytes of the innate immune system as well as naive, memory, and effector lymphocytes. The combinatorial diversity in responsiveness to chemokines ensures proper tissue distribution of distinct leucocyte subsets under normal and inflammatory/pathological conditions. Here, we discuss recent views on the role of chemokines in controlling tissue localisation of human memory T cells under steady state (non-inflamed) conditions. Emphasis is placed on a concept describing distinct subsets of memory T cells according to their primary residence in peripheral blood, secondary lymphoid tissues, or peripheral (extralymphoid) tissues.

Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8(+) T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8(+) T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor-beta was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8(+)CD25(-) T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin.

γδ T cells are inadequately defined both in terms of their migration potential and contribution to antimicrobial immunity. Here, we have examined the migration profile of human blood γδ T cells and related cell lines and correlated these findings with their distribution in secondary lymphoid tissues and their function in B-cell cocultures. We find that resting γδ T cells are characterized by an inflammatory migration program similar to cells of the innate immune system. However, T-cell receptor (TCR) triggering resulted in the rapid but transient induction of a lymph node (LN)-homing program, as evidenced by functional CCR7 expression and concomitant reduction in expression and function of CCR5 and, to a lesser degree, CCR2. Moreover, the LN-homing program was reflected by the presence of γδ T cells in gastrointestinal lymphoid tissues, notably in clusters within germinal centers of B-cell follicles. In line with these findings, VγVδ-TCR triggering resulted in prominent expression of essential B-cell costimulatory molecules, including CD40L, OX40, CD70, and ICOS. Furthermore, γδ T cells were shown to provide potent B-cell help during in vitro antibody production. Collectively, our findings agree with a role for γδ T cells in humoral immunity during the early phase of antimicrobial responses. (Blood. 2003; 102:3693-3701)

Introduction Hemolysis is considered a class effect and a rare adverse event that can occur following therapy with human normal immunoglobulin for intravenous administration [i.e., intravenous immunoglobulin (IVIG)]. Anti-A/B isoagglutinins (also referred to as isohemagglutinins) originating from donor plasma are present in polyvalent immunoglobulin G (IgG) products and are considered a probable risk factor for hemolysis. We hypothesized that, by excluding plasma from donors with high isoagglutinin titers, the final IVIG product would have a meaningful reduction in anti-A/B isoagglutinin titers. Methods A method for screening donor plasma for anti-A isoagglutinins using an automated indirect agglutination test (IAT) was developed. A cut-off for donor plasma exclusion was defined. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen® (CSL Behring, Berne, Switzerland; human 10% liquid IVIG). Anti-A/B isoagglutinin content in pooled plasma and final IVIG product was measured by IAT, direct agglutination test, and a flow cytometry-based assay [fluorescence-activated cell sorting (FACS) anti-A]. Results Screening of plasma from 705 donors identified 48 (6.8%) donors with high anti-A isoagglutinin titers in plasma (IAT agglutination score ≥2+ in a 1:200 pre-dilution). Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in pooled plasma, confirmed by a twofold anti-A isoagglutinin concentration reduction measured by FACS anti-A (1,352 vs. 2,467 µg/g IgG). When the same screening and exclusion were applied to industrial-scale plasma pools (resulting in the exclusion of plasma from 5% of donors), anti-A isoagglutinins were reduced by one titer step in the final IVIG product. Anti-B isoagglutinins were also reduced by one titer step, as many donors with high anti-A isoagglutinins also have high anti-B. Conclusion Reduction of anti-A/B isoagglutinin titers in IVIG products on an industrial scale is feasible through implementation of anti-A donor screening, which may reduce the risk of hemolysis following IVIG therapy. Electronic supplementary material The online version of this article (doi:10.1007/s13554-014-0016-2) contains supplementary material, which is available to authorized users.

Leukocyte traffic through secondary lymphoid tissues is finely tuned by chemokines. We have studied the functional properties of a human T cell subset marked by the expression of CXC chemokine receptor 5 (CXCR5). Memory but not naive T cells from tonsils are CXCR5+ and migrate in response to the B cell–attracting chemokine 1 (BCA-1), which is selectively expressed by reticular cells and blood vessels within B cell follicles. Tonsillar CXCR5+ T cells do not respond to other chemokines present in secondary lymphoid tissues, including secondary lymphoid tissue chemokine (SLC), EBV-induced molecule 1 ligand chemokine (ELC), and stromal cell–derived factor 1 (SDF-1). The involvement of tonsillar CXCR5+ T cells in humoral immune responses is suggested by their localization in the mantle and light zone germinal centers of B cell follicles and by the concomitant expression of activation and costimulatory markers, including CD69, HLA-DR, and inducible costimulator (ICOS). Peripheral blood CXCR5+ T cells also belong to the CD4+ memory T cell subset but, in contrast to tonsillar cells, are in a resting state and migrate weakly to chemokines. CXCR5+ T cells are very inefficient in the production of cytokines but potently induce antibody production during coculture with B cells. These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).

S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatography As Steps to Reduce Isoagglutinin Titers in IVIG Liane Hoefferer, Ibrahim El Menyawi, Brigitte Siani, Martin Imboden, Annette Gaida, Isabelle Glauser, Reinhard Bolli, Katharina Willimann, Sandra Wymann, A. Adriano Marques, Eleanora Widmer; CSL Behring, Berne, Switzerland. RATIONALE: Hemolysis is a rare but potentially serious complication of high-dose IVIG therapy. Isoagglutinins originating in donor plasma are believed to play a major role in these reactions, but host factors are also important. We determined the effects of excluding plasma from donors with high isoagglutinin titers from the pools used to prepare IVIG, and of specific anti-A/B immunoaffinity chromatography (IAC), on the anti-A and anti-B titers of IVIG products prepared using the process for Privigen/ Hizentra. METHODS: A high-throughput assay was used to identify plasma donors with the highest anti-A titers. The effects of addition of IAC with A/Btrisaccharide-coupled resins into the manufacturing process were also studied. Anti-A/B isoagglutinin titers in the resulting IVIG preparations were measured by indirect agglutination and flow cytometry. RESULTS: Exclusion of the 5% of plasma donors with the highest anti-A titers reduced both anti-A and anti-B isoagglutinins in the final product by approx. 50% (a single 2-fold dilution step, i.e. reduction of the titer from 1:16 to 1:8, and 1:8 to 1:4, respectively. N530 lots). Specific IAC reduced anti-A and anti-B titers by at least two 2-fold dilution steps in the final product (>80% reduction shown by flow cytometry on standard red cells), while the content of antibodies against common microbial antigens remained unchanged. CONCLUSIONS: Anti-A/B isoagglutinin reduction in IVIG products is feasible using screening and exclusion of a small percentage of high anti-A titer donors andwith specific IAC. These approachesmay reduce the risk of hemolysis in IgG therapy.

Human gammadelta T cells are considered to play a vital role in protective immunity through cytokine secretion and cytotoxic activity. We report that cells expressing the Vgamma2Vdelta2+-T cell receptor (Vdelta2+ T cells) also display principal characteristics of professional antigen-presenting cells such as dendritic cells. Thus, when activated, these cells efficiently processed and displayed antigens and provided co-stimulatory signals sufficient for strong induction of naïve alphabeta T cell proliferation and differentiation. We suggest that, upon microbial activation, Vdelta2+ T cells participate in the induction of adaptive immune responses and that these cells may be a useful tool in vaccine development and immunotherapy.

gamma delta T cells are inadequately defined both in terms of their migration potential and contribution to antimicrobial immunity. Here, we have examined the migration profile of human blood gamma delta T cells and related cell lines and correlated these findings with their distribution in secondary lymphoid tissues and their function in B-cell cocultures. We find that resting gamma delta T cells are characterized by an inflammatory migration program similar to cells of the innate immune system. However, T-cell receptor (TCR) triggering resulted in the rapid but transient induction of a lymph node (LN)-homing program, as evidenced by functional CCR7 expression and concomitant reduction in expression and function of CCR5 and, to a lesser degree, CCR2. Moreover, the LN-homing program was reflected by the presence of gamma delta T cells in gastrointestinal lymphoid tissues, notably in clusters within germinal centers of B-cell follicles. In line with these findings, V gamma V delta-TCR triggering resulted in prominent expression of essential B-cell costimulatory molecules, including CD40L, OX40, CD70, and ICOS. Furthermore, gamma delta T cells were shown to provide potent B-cell help during in vitro antibody production. Collectively, our findings agree with a role for gamma delta T cells in humoral immunity during the early phase of antimicrobial responses.

Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney–expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin–sensitive Ca2+ mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP–elevating agents prostaglandin E2 and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.

SummaryDendritic cells (DCs) are major constituents of peripheral tissues, where they control immunity to foreign and self-antigens. The process of continuous DC renewal under homeostatic conditions is largely undefined. Here, we demonstrate that CD14+ DC precursors, either derived from CD34+ hematopoietic progenitor cells or isolated from blood, were attracted by the chemokine CXCL14, which is constitutively produced in healthy skin and other epithelial tissues. In a tissue model we show that human epidermal equivalents profoundly affected CD14+ DC precursors, including their suprabasal positioning and survival as well as their differentiation into Langerhans cell-like cells with potent antigen-presentation functions. Our model assigns unprecedented roles to CXCL14 and epidermal tissue as attractant and niche of differentiation, respectively, in the renewal of Langerhans cells under steady-state conditions.