Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

Mast cell-derived IL-10 suppresses germinal center formation by affecting T follicular helper cell function.

The most prevalent cancer diagnosed in the world is sunlight-induced skin cancer. In addition to being a complete carcinogen, UV radiation, the causative agent of skin cancer, induces immune suppression. Because UV-induced immune suppression is a well-recognized risk factor for skin cancer induction, it is crucial to understand the mechanisms underlying UV-induced immune suppression. Mast cells, which have recently emerged as immune regulatory cells, are particularly important in UV-induced immune suppression. UV exposure does not induce immune suppression in mast cell-deficient mice. We report that UV irradiation blocks germinal center (GC) formation, Ab secretion, and T follicular helper (Tfh) cell function, in part by altering the expression of transcription factors BCL-6 and BLIMP-1. No suppression of GC formation, Tfh cell IL-21 expression, or Ab secretion was observed in UV-irradiated mast cell-deficient (Kit(W-sh/W-sh)) mice. When mast cell-deficient mice were reconstituted with wild type mast cells, immune suppression was restored. Reconstituting the mast cell-deficient mice with bone marrow-derived mast cells from IL-10-deficient mice failed to restore the ability of UV radiation to suppress GC formation. Our findings demonstrate a function for mast cells, suppression of Tfh cell production, GC formation, and Ab production in vivo.

Altered affinity maturation in primary response to (4-hydroxy-3-nitrophenyl) acetyl (NP) after autologous reconstitution of irradiated C57BL/6 mice.

Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the lambda1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by lambda1 positive antibodies but fail to produce high affinity NP-binding IgG1 antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.

Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines.

High levels of the Rel/NF-kappaB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer's patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB(-/-)) mice, in contrast to nfkb1(-/-), but similar to nfkb2(-/-) mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB(-/-) splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB(-/-) spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.

Role of TRAF2/GCK in melanoma sensitivity to UV-induced apoptosis.

Radiation resistance is a hallmark of human melanoma, and yet mechanisms underlying this resistance are not well understood. We recently established the role of ATF2 in this process, suggesting that stress kinases, which contribute to regulation of ATF2 stability and activity, play an important role in the acquisition of such resistance. Here we demonstrate that changes in the expression and respective activities of TRAF2/GCK occur during melanoma development and regulate its sensitivity to UV-induced apoptosis. Comparing early- and late-stage melanoma cells revealed low expression of TRAF2 and GCK in early-stage melanoma, which coincided with poor resistance to UV-induced, TNF-mediated apoptosis; forced expression of GCK alone or in combination with TRAF2 efficiently increased JNK and NF-kappaB activities, which coincided with increased protection against apoptosis. Conversely, forced expression of the dominant negative form of TRAF2 or GCK in late-stage melanoma cells reduced NF-kappaB activity and decreased Fas expression, resulting in a lower degree of UV-induced, Fas-mediated cell death. Our results illustrate a mechanism in which protection from, or promotion of, UV-induced melanoma cell death depends on the nature of the apoptotic cascade (TNF or Fas) and on the availability of TRAF2/GCK, whose expression increases during melanoma progression. Oncogene (2000) 19, 933 - 942.

Molecular and cellular basis of the altered immune response against arsonate in irradiated A/J mice autologously reconstituted.

The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.

A DNA repair abnormality specific for rearranged immunoglobulin variable genes in germinal center B cells.

The somatic hypermutation mechanism produces high-rate mutagenesis specifically targeted to rearranged immunoglobulin (Ig) variable (V) gene segments during the germinal center (GC) stage of B lymphocyte differentiation. The mechanism of this process remains uncertain, partly due to the lack of a direct assay for hypermutation activity. In this study, a gene-specific DNA repair assay was used to compare the rate and quality of DNA repair in the mantle zone (MZ) and GC B cells at rearranged and unrearranged Ig V genes. GC B cells were distinguished from MZ B cells by a retarded repair rate specific for rearranged Ig V genes. In addition, a unique feature of GC cells after DNA repair was the appearance of predominant mutations in rearranged Ig VH5 gene PCR products. These predominant mutations also occurred in natural mutants of VH5 genes. However, repair-associated mutations reflected, at least in part, "template-jumping" during amplification of the residually damaged genomic template. Overall, these findings reflect a repair abnormality associated with the hypermutation process by the criteria of sequence- and B cell stage-specificity. We conclude that locus-specific retardation of DNA repair is a component of the hypermutation mechanism. RFLP or SSCP analysis provides a simple assay to monitor this repair abnormality as a surrogate biochemical marker for hypermutation during B cell differentiation.