Protein tyrosine kinases and phosphatases cooperate to regulate normal immune cell function. We examined the role of PEST domain-enriched tyrosine phosphatase (PEP) in regulating T cell antigen-receptor function during thymocyte development and peripheral T cell differentiation. Although normal naïve T cell functions were retained in pep-deficient mice, effector/memory T cells demonstrated enhanced activation of Lck. In turn, this resulted in increased expansion and function of the effector/memory T cell pool, which was also associated with spontaneous development of germinal centers and elevated serum antibody levels. These results revealed a central role for PEP in negatively regulating specific aspects of T cell development and function.

Lymphotoxin (LT)α is expressed by activated T cells, especially CD4+ T helper type 1 cells, and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LTα-deficient (LTα−/−) mice. LTα−/− mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very low levels of IgG after immunization with sheep red blood cells. We show here that LTα-expressing B cells are essential for the recovery of primary, secondary, and memory humoral immune responses in LTα−/− mice. It is not necessary for T cells to express LTα to support these immune functions. Importantly, LTα-expressing B cells alone are essential and sufficient for the formation of FDC clusters. Once these clusters are formed by LTα-expressing B cells, then LTα-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

LTα-deficient (LTα−/−) mice show altered splenic microarchitecture. This includes loss of normal B cell–T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTα−/− mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTα expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTα−/− mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTα−/− mice, follicle structure and a strong IgG response were retained. These data indicate that LTα-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTα−/− mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTα could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTα−/− mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTα−/− BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTα supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.