The RG7/11.1 monoclonal antibody reacts with the Fc region of rat IgG2b. It is commonly used as a secondary antibody to detect rat IgG2b antibodies in various diagnostic applications.

INVIVOMAB ANTI-RAT IGG2B (CLONE: RG7/11.1)

Deppisch, N., et al. (2015). “Efficacy and Tolerability of a GD2-Directed Trifunctional Bispecific Antibody in a Preclinical Model: Subcutaneous Administration Is Superior to Intravenous Delivery.” Mol Cancer Ther 14(8): 1877-1883. PubMed

Trifunctional bispecific antibodies (trAb) are novel anticancer drugs that recruit and activate different types of immune effector cells at the targeted tumor. Thus, tumor cells are effectively eliminated and a long-lasting tumor-specific T-cell memory is induced. The trAb Ektomab is directed against human CD3 on T cells and the tumor-associated ganglioside GD2, which is an attractive target for immunotherapy of melanoma in humans. To optimize clinical applicability, we studied different application routes with respect to therapeutic efficacy and tolerability by using the surrogate trAb Surek (anti-GD2 x anti-murine CD3) and a murine melanoma engineered to express GD2. We show that subcutaneous injection of the trAb is superior to the intravenous delivery pathway, which is the standard application route for therapeutic antibodies. Despite lower plasma levels after subcutaneous administration, the same tumor-protective potential was observed in vivo compared with intravenous administration of Surek. However, subcutaneously delivered Surek showed better tolerability. This could be explained by a continuous release of the antibody leading to constant plasma levels and a delayed induction of proinflammatory cytokines. Importantly, the induction of counter-regulatory mechanisms was reduced after subcutaneous application. These findings are relevant for the clinical application of trifunctional bispecific antibodies and, possibly, also other immunoglobulin constructs. Mol Cancer Ther; 14(8); 1877-83. (c)2015 AACR.

Huang, G., et al. (2014). “Characterization of transfusion-elicited acute antibody-mediated rejection in a rat model of kidney transplantation.” Am J Transplant 14(5): 1061-1072. PubMed

Animal models of antibody-mediated rejection (ABMR) may provide important evidence supporting proof of concept. We elicited donor-specific antibodies (DSA) by transfusion of donor blood (Brown Norway RT1(n) ) into a complete mismatch recipient (Lewis RT1(l) ) 3 weeks prior to kidney transplantation. Sensitized recipients had increased anti-donor splenocyte IgG1, IgG2b and IgG2c DSA 1 week after transplantation. Histopathology was consistent with ABMR characterized by diffuse peritubular capillary C4d and moderate microvascular inflammation with peritubular capillaritis + glomerulitis > 2. Immunofluorescence studies of kidney allograft tissue demonstrated a greater CD68/CD3 ratio in sensitized animals, primarily of the M1 (pro-inflammatory) phenotype, consistent with cytokine gene analyses that demonstrated a predominant T helper (TH )1 (interferon-gamma, IL-2) profile. Immunoblot analyses confirmed the activation of the M1 macrophage phenotype as interferon regulatory factor 5, inducible nitric oxide synthase and phagocytic NADPH oxidase 2 were significantly up-regulated. Clinical biopsy samples in sensitized patients with acute ABMR confirmed the dominance of M1 macrophage phenotype in humans. Despite the absence of tubulitis, we were unable to exclude the effects of T cell-mediated rejection. These studies suggest that M1 macrophages and TH 1 cytokines play an important role in the pathogenesis of acute mixed rejection in sensitized allograft recipients.

Reich, B., et al. (2013). “Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.” Kidney Int 84(1): 78-89. PubMed

Collagen-producing bone marrow-derived cells (fibrocytes) have been detected in animal models and patients with fibrotic diseases. In vitro data suggest that they develop from monocytes with the help of accessory cells and profibrotic soluble factors. Using a mouse model of renal fibrosis, unilateral ureteral obstruction, we found the number of circulating fibrocytes was not reduced when monocytes were depleted with a monoclonal antibody against CCR2 or when CCR2-/- mice with very low numbers of circulating or splenic monocytes were analyzed. The absence of CCR2, however, interfered with migration of fibrocytes into the kidney. The phenotype of splenic and renal fibrocytes was very similar and distinct from classical monocytes as fibrocytes expressed no CD115, medium levels of CCR2, and high levels of CD11b and Ly-6G. Using a depleting monoclonal antibody against Ly-6G or bone marrow chimeric mice expressing the diphtheria toxin receptor under the control of CD11b, we could efficiently deplete fibrocytes from the kidney. Depletion of fibrocytes or reduced migration of fibrocytes into the kidney resulted in lower renal expression of collagen-I. Thus, fibrocytes develop outside the kidney independent of infiltrating monocytes and rely on CCR2 for migration into target organs.

Ohno, T., et al. (2011). “Paracrine IL-33 stimulation enhances lipopolysaccharide-mediated macrophage activation.” PLoS One 6(4): e18404. PubMed

BACKGROUND: IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.

Svensson, M., et al. (2010). “CD4+ T-cell localization to the large intestinal mucosa during Trichuris muris infection is mediated by G alpha i-coupled receptors but is CCR6- and CXCR3-independent.” Immunology 129(2): 257-267. PubMed

Infection of mice with the gastrointestinal nematode Trichuris muris represents a valuable tool to investigate and dissect intestinal immune responses. Resistant mouse strains respond to T. muris infection by mounting a T helper type 2 response. Previous results have shown that CD4(+) T cells play a critical role in protective immunity, and that CD4(+) T cells localize to the infected large intestinal mucosa to confer protection. Further, transfer of CD4(+) T cells from immune mice to immunodeficient SCID mice can prevent the development of a chronic infection. In the current study, we characterize the protective CD4(+) T cells, describe their chemokine receptor expression and explore the functional significance of these receptors in recruitment to the large intestinal mucosa post-T. muris infection. We show that the ability to mediate expulsion resides within a subpopulation of CD4(+) T cells marked by down-regulation of CD62L. These cells can be isolated from intestine-draining mesenteric lymph nodes (MLN) from day 14 post-infection, but are rare or absent in MLN before this and in spleen at all times post-infection. Among CD4(+) CD62L(low) MLN cells, the two most abundantly expressed chemokine receptors were CCR6 and CXCR3. We demonstrate for the first time that CD4(+) CD62L(low) T-cell migration to the large intestinal mucosa is dependent on the family of G alpha(i)-coupled receptors, to which chemokine receptors belong. CCR6 and CXCR3 were however dispensable for this process because neutralization of CCR6 and CXCR3 did not prevent CD4(+) CD62L(low) cell migration to the large intestinal mucosa during T. muris infection.

Handel, T. M., et al. (2008). “An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo.” J Leukoc Biol 84(4): 1101-1108. PubMed

We demonstrated recently that P8A-CCL2, a monomeric variant of the chemokine CCL2/MCP-1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate-mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA-sensitized mice. This anti-inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A-CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A-CCL2 and wild-type (WT) CCL2, ruling out the hypothesis that P8A-CCL2 disrupts the chemokine gradient through systemic exposure. P8A-CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A-CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A-CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP-1) from endothelial surfaces. Intravital microscopy studies indicate that P8A-CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization-deficient chemokines can exhibit anti-inflammatory properties in vivo and may represent new therapeutic modalities.

van den Brandt, J., et al. (2007). “Enhanced glucocorticoid receptor signaling in T cells impacts thymocyte apoptosis and adaptive immune responses.” Am J Pathol 170(3): 1041-1053. PubMed

To study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses.