Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 colocalize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca 2 þ -dependent cysteine protease, by upregulation of Ca 2 þ -permeable AMPA receptors generates carboxyterminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca 2 þ -impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

Depletion of CD4þ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4 þ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25 þ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8 þ cell depletion. CD4 þ cell depletion led to the proliferation of tumor-specific CD8 þ T cells in the draining lymph node and increased infiltration of PD-1into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies.

TDP-43 pathology in spinal motor neurons is a neuropathological hallmark of sporadic amyotrophic lateral sclerosis (ALS) and has recently been shown to be closely associated with the downregulation of an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) in the motor neurons of sporadic ALS patients. Because TDP-43 pathology is found more frequently in the brains of elderly patients, we investigated the age-related changes in the TDP-43 localization and ADAR2 activity in mouse motor neurons. We found that ADAR2 was developmentally upregulated, and its mRNA expression level was progressively decreased in the spinal cords of aged mice. Motor neurons normally exhibit nuclear ADAR2 and TDP-43 immunoreactivity, whereas fast fatigable motor neurons in aged mice demonstrated a loss of ADAR2 and abnormal TDP-43 localization. Importantly, these motor neurons expressed significant amounts of the Q/R site-unedited AMPA receptor subunit 2 (GluA2) mRNA. Because expression of unedited GluA2 has been demonstrated as a lethality-causing molecular abnormality observed in the motor neurons, these results suggest that age-related decreases in ADAR2 activity play a mechanistic role in aging and serve as one of risk factors for ALS.

<div>Abstract<p>Depletion of CD4⁺ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4⁺ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti–CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25⁺ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8⁺ cell depletion. CD4⁺ cell depletion led to the proliferation of tumor-specific CD8⁺ T cells in the draining lymph node and increased infiltration of PD-1⁺CD8⁺ T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti–PD-1 or anti–PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti–PD-1 or anti–PD-L1 mAb therapies. <i>Cancer Immunol Res; 3(6); 631–40. ©2015 AACR</i>.</p></div>

<p>Table S1. Antibodies for flow cytometry and immunohistostaining. Table S2. Primers and probes used in real-time PCR. Supplementary Figure S1. Generation of B16F10-ÃŽ?hLNGFR transfected cells. Supplementary Figure S2. Optimization of the timing and dose for the administration of anti-CD4 mAb monotherapy. Supplementary Figure S3. Anti-CD4 mAb treatment decreases numbers of CD4+ T cells and pDCs in the blood, lymph nodes, spleen and tumor. Supplementary Figure S4. Intravascular staining distinguishes the intravascular and parenchymal leukocyte fractions in tumor tissue. Supplementary Figure S5. Anti-CD4 mAb treatment enhances the killing activity of tumor-infiltrating CD8+ T cells. Supplementary Figure S6. Anti-CD4 mAb treatment systemically increases the number of CD8+ CD44hi PD-1+ T cells. Supplementary Figure S7. Anti-CD4 mAb treatment causes proliferation of tumor-specific CD8+ T cells. Supplementary Figure S8. Anti-CD4 mAb treatment enhances proliferation of tumor-specific CD8+ T cells in the dLN. Supplementary Figure S9. Anti-CD4 mAb treatment or combination treatment with anti-CD4 and anti-PD-1 or PD-L1 mAbs induces anti-tumor gene expression within the tumor.</p>

No abstract

Depletion of CD4+ cells in tumor-bearing mice has strong anti-tumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the anti-tumor effects of an anti-CD4 depleting monoclonal antibody (mAb) as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26 or LLC subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong anti-tumor effects that were superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, with a shift towards type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies. Citation Format: Satoru Ito, Kouji Matsushima, Satoshi Ueha, Shoji Yokochi, Yoshiro Ishiwata, Kosuke Hachiga, Haru Ogiwara, Krishant Chand, Takumi Nakajima. Robust anti-tumor effects of combined anti-CD4 depleting antibody and anti-PD-1/PD-L1 immune checkpoint antibody treatment in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 265. doi:10.1158/1538-7445.AM2015-265

<p>Table S1. Antibodies for flow cytometry and immunohistostaining. Table S2. Primers and probes used in real-time PCR. Supplementary Figure S1. Generation of B16F10-ÃŽ?hLNGFR transfected cells. Supplementary Figure S2. Optimization of the timing and dose for the administration of anti-CD4 mAb monotherapy. Supplementary Figure S3. Anti-CD4 mAb treatment decreases numbers of CD4+ T cells and pDCs in the blood, lymph nodes, spleen and tumor. Supplementary Figure S4. Intravascular staining distinguishes the intravascular and parenchymal leukocyte fractions in tumor tissue. Supplementary Figure S5. Anti-CD4 mAb treatment enhances the killing activity of tumor-infiltrating CD8+ T cells. Supplementary Figure S6. Anti-CD4 mAb treatment systemically increases the number of CD8+ CD44hi PD-1+ T cells. Supplementary Figure S7. Anti-CD4 mAb treatment causes proliferation of tumor-specific CD8+ T cells. Supplementary Figure S8. Anti-CD4 mAb treatment enhances proliferation of tumor-specific CD8+ T cells in the dLN. Supplementary Figure S9. Anti-CD4 mAb treatment or combination treatment with anti-CD4 and anti-PD-1 or PD-L1 mAbs induces anti-tumor gene expression within the tumor.</p>