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Central and peripheral tolerance mechanisms exist to prevent the adaptive immune system from reacting to self Ag. During T cell development, thymocytes migrate through the thymic architecture and undergo positive and negative selection. Weak recognition of peptides presented by cortical thymic epithelial cells induces positive selection. From there, thymocytes must survive negative selection largely mediated by dendritic cells in the medulla, although other potential APCs are present. Thymocytes in humans express MHC class I and II, and murine thymocytes express MHC class I, suggesting that they are capable of presenting Ag to other thymocytes. Melichar et al. (p. 1057) investigated whether developing thymocytes themselves could mediate negative selection. The investigators took advantage of a system in which cells can be exogenously added to thymic slices, allowing for the retention of thymic structure and chemokine signaling, which are crucial to T cell development. OTI TCR transgenic Rag2^−/− (OTItg) CD4⁺CD8⁺ thymocytes were placed onto nonselecting β2m^−/− thymic slices and wild-type (WT) thymocytes were added to serve as APCs. OTItg thymocytes only upregulated CD69 when the WT thymocytes had been preloaded with OVA peptide, but not with a low affinity OVA variant. The presence of OVA caused over three quarters of the OTItg cells to be deleted within 24 h, presumably through negative selection. Observation of direct interactions between cells with two-photon time-lapse microscopy revealed that 5% of the tracked OTItg thymocytes formed stable conjugate pairs with thymocyte APCs for the duration of the observation period. These thymocyte–thymocyte interactions were dependent on the presence of OVA peptide. Moreover, OTItg thymocytes in conjugate pairs displayed increased [Ca²⁺]_i compared with OTItg thymocytes not interacting with thymocyte APCs, indicative of TCR signaling. These results suggest a previously unknown role for thymocytes in thymic negative selection.

Elderly individuals, who are prone to more inflammatory immune responses, are vulnerable to respiratory bacterial infections. In this issue, Ghanem et al. (p. 1090) studied whether dietary supplementation with α-tocopherol (α-Toc, a form of vitamin E) could reduce morbidity and mortality specifically seen in aged mice following Streptococcus pneumoniae infection. Almost all two-year-old mice infected with a high dose of S. pneumoniae succumbed to infection within two days and had an abundance of neutrophils in their lungs. Younger two-month-old mice had lower levels of neutrophil recruitment and inflammatory cytokines in the lungs and over 90% were alive at this early time point. For four weeks, young and aged mice were fed either a control diet of 30 ppm α-Toc or a treatment diet containing 500 ppm α-Toc. The mice were then challenged with a low dose of S. pneumoniae. Overall, α-Toc treatment reversed the inflammatory phenotype seen in aged mice in response to S. pneumoniae infection. α-Toc–treated aged mice had a bacterial burden comparable to that of control and treated young mice and had decreased incidence of systemic spread compared with aged controls. Additionally, lung inflammatory cytokine levels as well as neutrophil airway invasion in treated aged mice resembled the levels seen in both groups of young mice. Experiments with cell lines and healthy human neutrophils revealed that α-Toc was capable of modulating pulmonary inflammation by altering adhesion molecule expression on neutrophils and epithelial cells, which may prevent neutrophil transmigration into the lungs. These results suggest that treatments like vitamin E supplementation may help protect elderly individuals from potentially life-threatening respiratory infections, in part by modifying neutrophil invasion into the airways.

During the establishment of central tolerance in the thymus, medullary thymic epithelial cells (mTECs) express a broad array of tissue-restricted self Ags (TRAs) through the process of promiscuous gene expression (pGE). The autoimmune regulator (AIRE) is known to play a role in this pGE, but much remains to be learned regarding how pGE occurs and how AIRE expression and activity are controlled. Because AIRE has been shown to act as part of a large multiprotein complex, Rattay et al. (p. 921) performed a yeast two-hybrid screen to identify AIRE binding partners that might be involved in the control of pGE. This screen identified the homeodomain-interacting protein kinase 2 (HIPK2) as an AIRE interacting factor. HIPK2 partially colocalized with AIRE in the nucleus and at nuclear bodies following cotransfection, as well as in mTECs in situ. HIPK2 could directly phosphorylate AIRE and, through a mechanism requiring its kinase activity, inhibit AIRE’s transactivation function. To further address the function of HIPK2 in the thymus, the authors generated conditional HIPK2 knockout mice (HIPK2^ko) with Hipk2 specifically deleted in all TECs. This deletion significantly reduced the numbers of thymic mTECs, but not cortical TECs. Analysis of differentially expressed genes in HIPK2^ko versus wild-type mTECs revealed that HIPK2 deficiency modified the expression of more genes in CD80^lo mTECs than in CD80^hi mTECs (which undergo pGE and have upregulated AIRE), and generally suppressed gene expression, which was unexpected. HIPK2 deficiency selectively affected the expression of genes encoding TRAs but, surprisingly, did not selectively affect AIRE-dependent genes. This study identifies HIPK2 as one of many players in the regulation of AIRE activity, pGE, and the resulting establishment of tolerance in the thymus, but a full understanding of its role awaits further investigation.

Transplanted allogeneic tissue can be rejected by host T cells through multiple mechanisms. Direct rejection involves host T cells recognizing donor MHC molecules, usually those present on donor APCs in draining lymph nodes, and is responsible for the acute rejection of skin and solid organ grafts. Indirect rejection occurs when T cells respond to donor peptides presented by host APCs, and mediates acute skin and chronic allograft rejection. In this issue, Kant et al. (p. 1364) examined the role that secondary lymphoid organs play in direct and indirect rejection of cardiac and skin allografts. The authors used alymphoplasia mice (aly/aly), which exhibit spleen defects, disorganized thymic structure, and no lymph nodes or Peyer’s patches. Splenectomy essentially rendered these aly/aly-spl^– mice devoid of secondary lymphoid organs. Aly/aly-spl^– mice tolerated cardiac transplants, which were rejected by wild-type (wt) mice within two weeks. Additionally, aly/aly-spl^– mice that had accepted an allogeneic heart 50 d earlier tolerated skin grafts from the same type of donor indefinitely. The mechanism of tolerance took at least 30 d to develop and was shown to be mediated through CD4⁺ T cells, but not necessarily CD4⁺CD25⁺ T regulatory cells. Interestingly, aly/aly-spl^– mice acutely rejected primary skin grafts, indicating that allorejection of skin does not require secondary lymphoid organs, even though the rejection was delayed in comparison with wt recipients. This rejection was demonstrated to be dependent on CD8⁺ T cells, but not CD4⁺ T cells or NK cells. In vitro assays with T cells from aly/aly mice that had rejected skin transplants showed that the rejection was likely mediated through the direct pathway, as stimulation with donor APCs caused the recipient T cells to secrete IL-4 and IL-10 and stimulation with host APCs loaded with donor peptides induced no response. Aly/aly-spl^– mice receiving a second skin graft three weeks after the rejection of a primary skin graft rejected the second graft more quickly, indicating that these mice can sustain alloreactive memory T cells even in the absence of secondary lymphoid organs. Taken together, these results indicate that both rejection and tolerance of allografts can be induced even in the absence of secondary lymphoid organs.

Chimeric Ag receptor (CAR)-transduced T (CAR-T) cells have emerged as promising tools in the fight against cancer. CAR-T cells have highly potent antitumor effects, but also have the potential for off-target toxicity against cells expressing their target Ags, even at low levels, in areas outside the tumor. To better understand CAR-T cell activity, Watanabe et al. (p. 911) constructed a CD20CAR and assessed the threshold target Ag density necessary for the activity of CD8⁺ T cells transduced with this receptor. The CD20CAR consisted of an anti-CD20–single-chain variable fragment connected to CD3ζ, a CD28 costimulatory domain, and a truncated version of the epidermal growth factor receptor, which was used for selection and measurement of transduction efficiency. Using as target cells a panel of CEM T lymphoblastoid cell lines that expressed CD20 at levels ranging from very low to high, the authors found that CD20CAR-T cells could efficiently lyse CD20-CEMs expressing levels of CD20 too low for anti-CD20 mAbs to effectively mediate complement-dependent cytotoxicity. The Ag threshold necessary for activation, proliferation, and cytokine production of CD20CAR-T cells was also quite low, albeit slightly higher than the threshold required for cytotoxicity. Intriguingly, CD20CAR-T cells also efficiently lysed primary tumor cells that had downregulated CD20 expression following anti-CD20 therapy. Taken together, these data reveal the impressive ability of CAR-T cells to detect and act against cells expressing very low levels of target Ag and thus suggest benefits and risks for therapeutic development of CD20CAR-T cells for use in patients with CD20-positive malignancies.