Lymph node (LN) architecture and chemokine expression facilitate encounters between APCs and naive T cells by guiding migration to the paracortex. This migration is supported by a three-dimensional (3D) network of T zone fibroblastic reticular cells (TRC). Tomei et al. (p. 4273) have engineered a 3D model of the T zone stromal network with cultured TRCs to better understand the effects of lymph flow on chemokine expression and cell trafficking. They developed immortalized TRC clones derived from mouse LNs that showed typical TRC morphology and cell marker expression. These TRCs formed 3D networks upon long-term culture on a composite polyurethane scaffold with a collagen matrix. Exposure of these cultures to slow interstitial flow induced TRCs to form channel-like structures and align with the underlying matrix and also caused increased expression of CCL21, an important chemokine for APC and T cell migration. CCL21 expression was low in 3D cultures before the application of fluid flow and fell rapidly upon flow disruption. The dependence of CCL21 expression on fluid flow in this 3D model suggests that increased lymph flow during inflammation may be critical to boosting TRC chemokine expression and initiating the first steps of the adaptive immune response in LNs.

Effective adaptive immune responses against respiratory viruses are dependent on T cell migration to sites of infection. Previous work has shown that migration is facilitated by CD8+ T cell expression of the CCR5 and CXCR3 chemokine receptors, and Kohlmeier et al. (p. 4378) now show that CD4+ T cell migration is also guided by CXCR3, but not CCR5. They observed that the majority of virus-specific CD4+ T cells isolated from the lungs of mice infected with Sendai virus expressed CXCR3 and CCR5. No difference in virus-specific CD4+ T cell migration was observed in Ccr5−/−, Cxcr3−/−, or double knockout mice compared with wild-type mice. The contribution of these chemokine receptors to CD4+ T cell migration was revealed in mixed bone marrow chimeras created from congenic wild-type and chemokine receptor-deficient mice. Virus-specific CD4+ T cells proliferated in both wild-type and chemokine receptor-deficient chimeras, but CD4+ T cell migration to the lung was significantly decreased in Cxcr3−/− chimeric mice. Migration was not affected by Ccr5 deficiency, nor was it worsened in Ccr5−/− × Cxcr3−/− mice. Chemokine receptor deficiency did not affect migration to mediastinal lymph nodes and did not inhibit recruitment of NK or NKT cells to the lungs. Taken together, these data demonstrate that CXCR3 is vital to CD4+ T cell recruitment to the lungs during viral infection.

Several type 1 diabetes (T1D)-associated autoantigens have been developed for peptide-based therapies in nonobese diabetic (NOD) mice. The limited effectiveness of these therapies led Li et al. (p. 4809) to develop an alternative approach using peptide-MHC II-Ig fusion proteins carrying peptides from the pancreatic β cell Ag glutamic acid decarboxylase 65 (GAD65). Soluble recombinant forms of I-Ag7 MHC II-Ig dimers were covalently linked to one of two different GAD65 peptides or to a nonself peptide from hen egg lysozyme (sI-Ag7-GADp217, sI-Ag7-GADp290, or sI-Ag7-HEL). Intravenous administration of sI-Ag7-GADp217 or sI-Ag7-GADp290, but not sI-Ag7-HEL, suppressed autoimmune islet damage and prevented TID in late phase prediabetic NOD mice. Adaptive regulatory CD4+ T cells (aTreg) expressing IL-10 were responsible for suppressing autoreactive T cells, and FoxP3+ Treg also proliferated following treatment with GAD65-bearing MHC II-Ig molecules. IL-10-expressing aTreg suppressed proinsulin and insulin B chain-specific pathogenic T cells in addition to GAD65-specific T cells, suggesting evidence of epitope spreading. The successful suppression of TID in prediabetic NOD mice with this novel molecular approach restores hope for peptide-based immunotherapies that target diabetes and other T cell-mediated autoimmune diseases.

Antibody diversity is generated during Ig gene transcription through somatic hypermutation and gene conversion of rearranged Ig variable regions. Activation-induced deaminase (AID) is a key enzyme for initiating these diversification processes in B cells, and Ordinario et al. (p. 4545) used single-cell imaging to show that AID activity in chicken B cells occurs primarily during the G1 phase of the cell cycle. AID localized to the nucleus during G1 phase as determined by imaging chicken bursal lymphoma DT40 cells transfected with a yellow fluorescence protein-tagged form of chicken AID (AID-YFP). Nuclear AID-YFP levels fell during S phase through AID ubiquitination and subsequent proteolysis. Repair enzymes and transcription factors were expected to arrive at the sites of AID attack to repair DNA damage, and this was confirmed in DT40 cells that expressed a polymerized lactose operator-tagged form of the rearranged λR gene. The repair polymerase Polη and multifunctional factor MRE11/RAD50/NBS1 both colocalized with λR, and Polη/λR colocalization occurred primarily during G1 phase. Overall, these results indicate that Ig gene conversion takes place mainly during G1 phase, likely protecting DNA from inappropriate AID attack during DNA replication.

The MHC I Ag presentation pathway is essential for generating specific CD8+ T cells that target microbes or tumors, but such responses must be restrained to avoid autoimmunity. In this issue, two debated aspects of MHC I Ag presentation have been explored. In the first article, Ishizuka et al. (p. 4337) estimated the frequency of T cell cross-reactivity to unrelated peptides (fun). An accurate determination of fun has been limited by earlier approaches that used small or biologically less relevant peptide libraries. Cross-reactivity was measured using CD8+ T cell clones with TCRs specific to antiself or antiviral peptides and a large library of MHC I-restricted (H2-Kb, H2-Db, or HLA-A*0201) synthetic peptides derived from viral and bacterial open reading frames. A single authentic TCR-peptide MHC I (TCR-pMHC) cross-reaction was detected between an antiself TCR and a poxvirus peptide among almost 30,000 TCR-pMHC interactions. Virus-specific or alloreactive polyclonal CD8+ T cells did not show any measurable cross-reactivity with this peptide library, thus supporting observations with the T cell clones that estimated fun to be rare. Furthermore, in vitro-generated memory-like T cell clones were no more sensitive to cross-reactive peptides than naive T cells. These results, which were further supported by empirical calculations, estimated fun to be infrequent (∼1/30,000) in the context of CD8+ TCRs and peptide-MHC I complexes and confirmed earlier estimates of CD8+ T cell cross-reactivity to viral and self Ags.

In the second article, Lev et al. (p. 4205) addressed the debated role of glucose regulated protein 94 (GRP94) in the cross-priming of CD8+ T cells by dendritic cells (DCs) during viral infection. GRP94 is an endoplasmic reticulum-resident chaperone that has been suggested to be a cross-priming facilitator with evidence that it can bind peptides and be taken up by DCs. In this study, small interfering RNA-mediated silencing of GRP94 expression did not affect MHC I expression or Ag processing or presentation in virally infected cells. GRP94-deficient cells did not show any abnormalities in morphology, cell division, or expression of other chaperones. Strikingly, in vivo cross-priming of viral Ag-specific CD8+ T cells by transfected or virally infected cells was not altered by GRP94 silencing. Taken together, these results do not support an essential role for GRP94 in Ag presentation by DCs through direct- or cross-priming mechanisms. These findings may also provide a new perspective on the limited effectiveness of GRP94 in human cancer immunotherapy trials. Overall, these papers provide new insights and limits into the fundamental features of MHC I presentation.

Recent studies suggest that aluminum salt vaccine adjuvants (alum) have innate stimulatory properties that require the Nod-like receptor (NLR) family member NLRP3 inflammasome for Th2-polarized adaptive immune responses. In this issue, McKee et al. (p. 4403) challenged this model through a comprehensive study of the earliest immune responses initiated by alum. Intraperitoneal alum injection induced rapid peritoneal infiltration of eosinophils, neutrophils, monocytes, dendritic cells (DCs), NK cells, and NKT cells. Eosinophils were found to be the key IL-4-expressing cells in the peritoneal cavity. Macrophages and mast cells (MCs) were identified as alum sensors because IL-5- and histamine-mediated eosinophil recruitment and IL-1β production were dependent on both of these cell types. Surprisingly, eosinophils, macrophages, and MCs were dispensable for Ag-specific B and T cell responses in mice immunized with alum and the experimental Ag OVA. Caspase-1 was not essential for inflammatory cell infiltration, and adaptive immune responses were similar for caspase-1- or NLRP3-deficient mice compared with wild-type mice following alum/OVA immunization. These data show that alum can be sensed by macrophages and MCs, thus causing inflammasome activation and inflammatory cell infiltration, but these initial responses are not needed to mediate Th2-polarized adaptive immune responses.

Adaptive immune responses and ischemia/reperfusion (I/R) injury have been identified as factors causing allograft rejection. Activation of TLR/IL-1R signaling pathways appears to be essential for these responses, but TLR responses can be suppressed by endogenous negative regulators. Noris et al. (p. 4249) have identified Toll-IL-1R 8 (TIR8), a negative regulator of TLR4 and IL-1R signaling, as a critical molecule for kidney allograft acceptance. Spontaneous graft acceptance has been observed previously and was confirmed following transplantation of kidneys from C57BL/6 (Tir8+/+) mice to mismatched DBA/2 mice, but kidney grafts were rejected rapidly in DBA/2 mice receiving kidneys from Tir8−/− mice. Compared with Tir8+/+, Tir8−/− kidneys had greater signs of inflammation due to I/R injury, including macrophage and neutrophil infiltration, as well as enhanced maturation and proliferation of tissue-resident dendritic cell (DC) precursors. Tir8−/− kidney recipients had diminished regulatory T cell activity and an increased frequency of antidonor T cell responses compared with Tir8+/+ recipients. These responses may be caused by an increased allostimulatory capacity of Tir8−/− DC precursors. Thus, TIR8 appears to be a fundamental regulator of kidney allograft acceptance and has therapeutic potential for improvement of graft acceptance.

Summaries written by Christiana N. Fogg, Ph.D.