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Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

This file contains the following figures: Supplemental Figure 1. The return of host lymphocytes is affected by the type of donor CD8+ T cells. Supplemental Figure 2. Return of myeloid cells after CTX leads to more rapid host cell reconstitution than TBI-containing regimens. Supplemental Figure 3. CTX and TBI differentially affect host T cells and NK cells. Supplemental Figure 4. Donor CD8+ T cells transferred into a TBI-conditioned host maintain functional efficacy. Supplemental Figure 5. ALT-803 (IL-15/IL-15Ra complexes) augment the persistence of Tc1 cells in irradiated hosts.

The present study aims to study the effect of vitamin D (Vit D) or / and coconut oil (Coc) on the splenic histological changes of diabetic adult mice induced by STZ. The mice were divided into 7 groups, and the experimental duration was 4 weeks. GpI: control group without any treatments; GpII&GpIII: non-diabetic groups orally received Vit D in a dose of 500 IU (6.25 ml)/kg b.w/d or Coc in a dose of 7.5 ml /kg b.w/d; GpIV: diabetic group injected i.p. with a single dose of STZ (200 mg/kg bw); Gps V,VI &VII: administration of Vit D or Coc or both together to diabetic group. The results recorded: GpII&GpIII no significant changes in the blood glucose (BG), insulin&splenic weight levels. GpIV had a significant increase in BG, significant decrease in insulin & splenic weight values. GpV recorded a modest decrease in BG, and a modest rise in insulin & a marked splenic weight atrophy; while GpVI or GpVII registered a marked decrease in BG, a rise in insulin levels & a marked splenic weight increment. Histologically, the splenic sections of control or non-diabetic mice received either Vit D or Coc demonstrated normal structure of the splenocytes. GpIV showed great numbers of giant cells, disarrangement and interference of red and white pulps, and dilated congested blood vessels. Little improvement was seen in GpV while GpVI or GpVII showed a marked improvement in the splenic tissues. In conclusion, diabetic mice received either Coc or co-dministered with Vit D demonstrated strong anti-hyperglycemic effects to recovery the glucose and insulin rates to typical levels, and restored the splenic weight and histological architecture to normal status than those given Vit D alone.

Introduction:Leukopenia is one of the major side effects of myelosuppressive chemotherapy such as cyclophosphamide (CTX). We and others have used CTX either alone or in combination with G-CSF for the mobilization of hematopoietic stem cells (HSCs). This mobilization can induce expansion of myeloid cells with immunosuppressive phenotype. In this pilot study, we aimed to test whether bone marrow lysate (BML)/CTX, a rich source of growth factors, can lower the expansion of myeloid cells with immunosuppressive phenotypes in tumor-bearing mice without interfering with the anti-tumor effects of CTX or with the mobilization of HSCs.Methods:Female CD1 mice were treated on day 0 with an i.p. injection of Ehrlich ascites carcinoma (EAC). On day 7, the mice were i.p. injected with CTX followed by s.c. injection of G-CSF for 5 consecutive days, single s.c. injection of BML/PBS or BML/CTX or single i.v. injection of BMC/PBS or BMC/CTX.Results:Treatment of EAC-bearing mice with BML/PBS or BML/CTX did not interfere with the anti-tumor effect of CTX. EAC increased the numbers of immature polymorphonuclear cells (iPMN; neutrophils) in both blood and spleen. Treatment of EAC-bearing mice with CTX further increased the numbers of these cells, which were decreased upon treatment with BML/CTX. Treatment with BML/PBS or BML/CTX increased the numbers of stem cells (C.Kit+Sca-1+) in BM; the effect of BML/CTX was higher, but with no significant effect on the numbers of HSCs. Future studies are needed to analyze the molecular components in BM lysate and to determine the underlying mechanisms.

In a series of our preclinical studies, we have reported that conditioning of α/β CD8We aimed in this study to use IL-12 to enhance the expansion and cytotoxic functions of ZOL-expanded Vγ9Peripheral blood mononuclear cells (PBMCs) were separated from healthy donors and stage II breast cancer patients. PBMCs (1 × 10In healthy subjects, the addition of IL-12 to ZOL/IL2-stimulated PBMCs increased the expansion and the cytotoxic activity of Vγ9The addition of IL12 at the start of the expansion protocol can enhance the activity of γδ T cells which might be mediated in part by the activation of αβ T cells.

Background: Anti-cancer adoptive T cell therapy has shown significant anti-tumor responses in cancer patients. This therapy is based on in vitro activation of autologous T cells harvested from a cancer patient and infusing them back through blood. The efficacy of adoptively transferred T cells depends on the in vitro conditioning regimen in particular the type of used cytokines. Aim: This study aimed to use toll like receptors (TLRs) ligands (TLRLs) to condition T cells in vitro as a novel approach instead of cytokines. Methods: Spleen cells were harvested from TCR transgenic C57BL/6 pmel-1 mice, in which CD8+ T cells are engineered to recognize melanoma MHC class-I peptides.Unfractionated splenocytes were cultured for 24 hours in vitro with media or melaoma peptide plus or minus IL-12 (10ng/ml), poly(I:C) (TLR3L; 25ug/ml) or CpG (TLR9L; 10ug/ml). Then, activation, proliferation and cytokine production of the cultured cells were assessed. In another set of experiments, the cultured cells were harvested and infused into syngeneic B6 mice followed by vaccination to evaluate their antigen-specific expansion and contraction. Results: Conditioning of donor splenocytes in vitro with the TLR3L or TLR9L during antigen stimulation increased the antigen specific activation, proliferation, and cytokine production. Interestingly, in vitro treatment of the unfractionated splenocytes with TLR9L enhanced the activation and proliferation of B cells regardless antigen stimulation. Adoptive transfer of TLRL-conditioned peptide stimulated CD8+ T cells into naïve mice showed better survival and higher expansion in response to concomitant vaccination with peptide. Interestingly, in vitro conditioning of CD8+ T cells with TLR9L and in vivo conditioning with TLR3L resulted in the best antigen specific expansion of these cells upon their adoptive transfer into recipient mice. Conclusion: These results show that provision of CD8+ T cells in vitro and in vivo with certain TLR agonists can markedly enhance their antigen specific responses upon their adoptive transfer, opening a potential application of this approach in anti-cancer adoptive immunotherapy.

Hepatitis C virus (HCV) is a blood-borne virus. Given that mosquitoes can take blood meals from HCV patients, we aimed to test whether HCV in the blood meal can induce alterations in the biology of Culex pipiens. To address this aim, Cx. pipiens females were fed HCV-negative blood from healthy individuals or HCV-positive fresh blood samples harvested from viremic HCV patients. Replication of HCV in mosquitoes was confirmed by negative strand-specific RT-PCR and sequencing of RNA extracted from the mosquito bodies 7 days post-feeding. In addition, several parameters that determine the fitness of the mosquitoes were measured. Virus acquisition was associated with alterations in the architecture of the gut microvilli and the immune response, indicated by an increase in phenol oxidase activity. Interestingly, the mosquitoes that were fed the HCV-positive blood meal showed shorter median longevity (8 days) and laid fewer eggs than the control mosquitoes. Furthermore, the offspring of females fed the HCV-positive blood meal demonstrated a lower emergence rate than the controls. In sum, the results indicate that feeding on HCV by Cx. pipiens decreases fitness, which may, in turn, affect its potential as a vector.