The reduced numbers of mature T cells and the impaired TCR signaling in thymocytes and splenic CD4+ T cells in the absence of RhoH suggested perturbed T cell dependent humoral immunity.
The primary immune response is triggered when a naïve T cell upon encounter with an antigen presented by a professional antigen presenting cell (APC), such as a DC, macrophage or B cell in the secondary lymphoid organ, is activated and becomes an effector T cell. DCs
are the most potent activators of T cells. CD8+ T cells recognize the processed antigen in the context of MHC class I on the APC and differentiate to cytotoxic T cells that can kill infected cells by releasing cytotoxins stored in special granula. CD4+ T cells bind antigens in the context of MHC class II and differentiate into the helper Th1 or Th2 cells. Th1 cells stimulate germ-killing activity of macrophages, while Th2 cells stimulate B cells to secrete antibodies. The effector T cells proliferate and either migrate to the site of the infection (as CD8+ T cells and Th1 cells) to remove pathogens or remain in the secondary lymphoid organs to activate B cells to produce antibodies (as Th2 cells). In the early phase of the immune response IgM is synthetized. This process is T cell independent. Later on, with the T cell help, class switching is initiated in B cells to produce IgG1, IgG2a, IgG2b, IgG3, IgE or IgA that have different functions in the immune response (Janeway et al., 2001). The class switch to a certain isotype depends on the cytokines produced by Th1 and Th2 cells. Th1 cells secrete IFN-γ, IL-2, TNF- α and TNF-β, favouring the class switch to IgG2a, IgG2b, IgG3 and IgA. Th2 cells generate IL-4, IL-5, IL-6 and IL-13, stimulating the production of IgG1 and IgE (Szabo et al., 2003). Thereby, these cells direct ongoing immune response through the secretion of cytokines. In germinal centres of lymphoid follicles, B cells activate SHM leading to affinity maturation of antibodies which can recognize the pathogens with higher affinity. During secondary immune response memory T and B cells, which develop at the time of the primary immune response, can counteract the pathogens with more efficiency.
To determine whether the loss of RhoH affects antibody production, we immunized control and RhoH-null mice with the T cell dependent (TD) antigen nitrophenyl-chicken-γ-globulin (NP20-CG) and studied the development of the immune response. Immunization with NP20-
CG will lead mainly to a Th2 response, resulting in a production of IgG1 and to a lesser degree to a production of IgG2a and IgG3, characterizing a Th1 response.
RhoH was reported to be expressed stronger in Th1 subset of CD4+ T cells than in Th2 cells, another subset of CD4+ helper T cells, indicating that in the absence of RhoH, there might be a skewing of helper CD4+ T cells towards Th2 subset in the course of the immune response. The dominance of Th2 cells might lead to a shift in the secretion of cytokines towards Th2 type that in turn would induce a skewing of antibody isotypes towards IgG1. Therefore we compared the serum concentrations of NP20-CG specific antibodies of IgG2a and IgG3
isotypes, which prevail in a Th1 response, with the concentration of antibodies of the IgG1 isotype, which is indicative of a Th2 response, at different time points after immunization. The NP20-CG specific titers of antibodies were determined by means of ELISA.
Figure 46. Impaired T cell dependent production of IgG1 and Igλ antibodies in RhoH-deficient mice. 2
month old control and RhoH-deficient mice were intraperitoneally injected with NP20-CG and boosted on day 42. Production of NP20-CG specific IgM (A) was determined by ELISA after 7, 14 and 21 days. Serum levels of NP20-CG specific IgG2a (C) and IgG3 (D) were measured after 28, 42, 49 and 56 days. Response of NP20-CG specific IgG1 (B), Igλ (E) and Igκ (F) antibodies was examined after 7, 14, 21, 28, 42, 49 and 56 days. [days 1- 42: n=5/5; days 49-56: n=5/4); *: p<0.05; **: p<0.01; y axis is logarithmic].
The production of the IgM antibody, which is the first isotype to emerge in a humoral immune response and which does not require T cell help, was not changed after 7, 14 and 21 days in RhoH-null mice compared to controls (Figure 46A). IgM molecules form pentamers with 10 binding sites and therefore display high stickiness which could explain the relatively high levels of NP20-CG specific IgM titer before the immunization in both RhoH-mutant and
control mice (Figure 46A).
IgG production is dependent on T cell help. Before the NP20-CG injection, IgG1 antibodies
were below the detection threshold in RhoH-null and control mice (Figure 46B). During the primary response (day 1 to day 28) the IgG1 titer was induced and maintained at higher levels than IgM titers in control mice. At day 42 the titer fell and began to increase again during memory response in control mice (Figure 46B). In RhoH-deficient mice, the IgG1 antibody production followed the IgG1 response in control mice. However, the levels of IgG1 antibodies during initial response as well as after the boost immunization were reduced, demonstrating that the Th2 type and the memory response were affected (Figure 46B).
The titers of IgG2a and IgG3 were determined at the end of the initial immunization at day 28 and during the booster response at the days 42, 49 and 56. The production of IgG2a and IgG3 is T cell dependent. IgG3 levels were low at all time points and exhibited no significant differences between RhoH-null and control mice (Figure 46C, D). The titers of IgG2a antibodies increased in the course of the memory response and were similar in RhoH-mutant and control mice (Figure 46C, D). In contrast to our expectations, these data revealed no skewing towards a Th2 type response.
Next, we tested the production of immunoglobulin light chain Igκ and Igλ. The distribution of κ and λ light chains paired with the heavy chain depends on the mouse strain and the carrier protein bound to the hapten. The immunization with NP20-CG elicits the production of λ chain
over κ chain. In the mice on the mixed background C57BL6/129Sv the NP20-CG immunogen
induced predominantly Igλ antibodies. After the first NP20-CG injection Igλ titers increased
and were maintained at high levels during the primary reaction (Figure 46E). The Igλ titers decreased in the time from day 28 to day 42 and rose again during booster response in controls. The levels of Igλ were reduced during primary and memory response in the absence of RhoH (Figure 46E). The development of Igκ antibody response was similar to Igλ, however the Igκ levels were lower than those of Igλ. During the primary response, the Igκ titers were not different between control and RhoH-deficient mice, while during secondary response they were slightly, although not significantly, reduced in the absence of RhoH (Figure 46F).
Taken together, the T cell independent induction of IgM antibodies was not affected by the loss of RhoH, whereas the T cell dependent class switching to IgG1 and Igλ was impaired in the absence of RhoH. No indication of skewing to Th2 type response was observed.