Higher Numbers Of CD8 Research Articles

Novel culture media enhances mononuclear cells from patients with chronic limb-threatening ischemia to increase vasculogenesis and anti-inflammatory effect

BackgroundQuality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function.MethodsPatients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017–December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media.ResultsThirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p < 0.0001). The percentage of CD206+ cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 19.31 ± 11.42% and 4.40 ± 2.54%, respectively (p < 0.0001). The percentage of inactive population of T regulatory cells (CD4+CD25+CD127+ cells) in MNCs cultured in standard culture media and in MNCs cultured in QQ culture media was 14.5 ± 10.68% and 1.84 ± 1.37%, respectively (p < 0.0001). The total number of colony-forming units from MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 8.86 ± 8.35 of 2 × 105 cells/dish, and 0.58 ± 1.05 of 2 × 105 cells/dish, respectively (p < 0.0001). The mean intensity of Dil-Ac-LDL uptake that incorporated into the HUVEC forming tube was 1.37 ± 0.88 in MNCs cultured in QQ culture media, and 0.78 ± 0.41 in MNCs cultured in standard culture media. (p < 0.0003).ConclusionsMNCs from CLTI patients that were cultured in QQ culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media.

Inflammation-Related Biomarkers Are Associated with Heart Failure Severity and Poor Clinical Outcomes in Patients with Non-Ischemic Dilated Cardiomyopathy.

Inflammation-related biomarkers are associated with clinical outcomes in mixed-etiology chronic heart failure populations. Inflammation-related markers tend to be higher in ischemic than in non-ischemic dilated cardiomyopathy (NI-DCM) patients, which might impact their prognostic performance in NI-DCM patients. Therefore, we aimed to assess the association of inflammation-related biomarkers with heart failure severity parameters and adverse cardiac events in a pure NI-DCM patient cohort. Fifty-seven patients with NI-DCM underwent endomyocardial biopsy. Biopsies were evaluated by immunohistochemistry for CD3+, CD45ro+, CD68+, CD4+, CD54+, and HLA-DR+ cells. Blood samples were tested for high-sensitivity C-reactive protein (hs-CRP), interleukin-6, tumor necrosis factor-α (TNF-α), soluble urokinase-type plasminogen activator receptor and adiponectin. During a five-year follow-up, twenty-seven patients experienced at least one composite adverse cardiac event: left ventricle assist device implantation, heart transplantation or death. Interleukin-6, TNF-α and adiponectin correlated with heart failure severity parameters. Patients with higher levels of interleukin-6, TNF-α, adiponectin or hs-CRP, or a higher number of CD3+ or CD45ro+ cells, had lower survival rates. Interleukin-6, adiponectin, and CD45ro+ cells were independently associated with poor clinical outcomes. All patients who had interleukin-6, TNF-α and adiponectin concentrations above the threshold experienced an adverse cardiac event. Therefore, a combination of these cytokines can identify high-risk NI-DCM patients.

High-dose IL-2/CD25 fusion protein amplifies vaccine-induced CD4+ and CD8+ neoantigen-specific T cells to promote antitumor immunity

BackgroundImmunization with tumor neoantigens is a promising vaccine approach to promote antitumor immunity due to their high immunogenicity, lack of expression in normal tissue, and preferential induction of tumor neoantigen-specific...

Environmental Air Pollutants Inhaled during Pregnancy Are Associated with Altered Cord Blood Immune Cell Profiles.

Air pollution exposure during pregnancy may be a risk factor for altered immune maturation in the offspring. We investigated the association between ambient air pollutants during pregnancy and cell populations in cord blood from babies born to mothers with asthma enrolled in the Breathing for Life Trial. For each patient (n = 91), daily mean ambient air pollutant levels were extracted during their entire pregnancy for sulfur dioxide (SO2), nitric oxide, nitrogen dioxide, carbon monoxide, ozone, particulate matter <10 μm (PM10) or <2.5 μm (PM2.5), humidity, and temperature. Ninety-one cord blood samples were collected, stained, and assessed using fluorescence-activated cell sorting (FACS). Principal Component (PC) analyses of both air pollutants and cell types with linear regression were employed to define associations. Considering risk factors and correlations between PCs, only one PC from air pollutants and two from cell types were statistically significant. PCs from air pollutants were characterized by higher PM2.5 and lower SO2 levels. PCs from cell types were characterized by high numbers of CD8 T cells, low numbers of CD4 T cells, and by high numbers of plasmacytoid dendritic cells (pDC) and low numbers of myeloid DCs (mDCs). PM2.5 levels during pregnancy were significantly associated with high numbers of pDCs (p = 0.006), and SO2 with high numbers of CD8 T cells (p = 0.002) and low numbers of CD4 T cells (p = 0.011) and mDCs (p = 4.43 × 10−6) in cord blood. These data suggest that ambient SO2 and PM2.5 exposure are associated with shifts in cord blood cell types that are known to play significant roles in inflammatory respiratory disease in childhood.

Abstract 26: Association of aneuploidy score with clinical outcomes to immunotherapy in NSCLC

Abstract Introduction: Cancer aneuploidy, an unbalanced number of chromosomes, is associated with somatic mutation rate, expression of proliferative genes, and altered immune signaling. Whether aneuploidy impacts clinical outcomes to immune checkpoint inhibitors (ICIs) in NSCLC is unknown. Methods: In NSCLCs which underwent targeted next-generation sequencing (NGS), we retrospectively analyzed the aneuploidy score (AS), defined as the sum of the number of altered chromosome arms, among patients treated with immune checkpoint inhibitors. An unbiased recursive partitioning (URP) algorithm was used to investigate an optimal AS cut-off with respect to objective response rate (ORR). Multiplexed immunofluorescence (mIF) to quantify CD8+, FOXP3+, PD-1+ immune cells, and PD-L1 was performed to determine differences in tumor immune cells subsets according to AS cut-off. Results: Among 279 patients with NSCLC treated with ICIs, the median AS was 6 (range 0 to 23). The AS was significantly lower among patients with a partial response to ICI compared to those with stable or progressive disease (4 vs 7, P=0.004). An unbiased recursive partitioning analysis identified an AS of 2 as the strongest discriminator of objective response to ICI. Compared to patients with an AS &amp;gt;2 (N= 207, 74.2%), patients with AS ≤2 (N=72, 25.8%) had a significantly higher overall response rate (ORR 43.0% vs 19.8%, P&amp;lt;0.001), a significantly longer median progression free survival (mPFS 6.2 months vs 2.9 months, HR: 0.70 [95% CI: 0.52-0.94], P=0.02), and a significantly longer median overall survival (mOS 19.8 months versus 13.8 months, HR: 0.66 [95% CI: 0.47-0.94], P=0.02) to treatment with ICIs. After adjusting for other variables such as performance status, presence of oncogenic driver mutation, PD-L1 expression, tumor mutational burden, and line of treatment, AS was significantly associated with improved mPFS (HR: 0.72 [95% CI: 0.52-0.99], P=0.04) and mOS (HR: 0.64 [95% CI: 0.44-0.94], P=0.02). Among 179 NSCLCs profiled by multiplex immunofluorescence, compared to cancers with an AS &amp;gt;2, those with low aneuploidy (AS ≤2) had significantly higher numbers of CD8+, FOXP3+, PD-1+ immune cells, and PD-1+ CD8+ T cells, both intratumorally and when looking at the total numbers of cells within the tumor and the tumor-stroma interface. There was no significant difference in PD-L1 expression levels or tumor mutational burden on tumor cells or on immune cells according to aneuploidy score. Conclusion: NSCLCs with low aneuploidy have a distinct immune microenvironment and more favorable outcomes to ICIs. Citation Format: João Victor Alessi, Biagio Ricciuti, Yvonne Y. Li, Hersh Gupta, Giuseppe Lamberti, Gonzalo Recondo, Mizuki Nishino, Lynette M. Sholl, Andrew D. Cherniak, James Lindsay, Bijaya Sharma, Kathleen Pfaff, Kristen Felt, Scott Rodig, Mark M. Awad. Association of aneuploidy score with clinical outcomes to immunotherapy in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 26.

Baseline Biomarkers Influencing Clinical Outcomes in Adults with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (R/R ALL) Treated with Blinatumomab Versus Standard-of-Care Chemotherapy (SOC) from a Randomized Phase 3 Study

Introduction: Blinatumomab, an anti-CD3/CD19 bispecific T-cell engager antibody construct, showed antileukemic activity and significantly longer overall survival (OS) vs SOC in adults with advanced Philadelphia chromosome-negative R/R ALL (median OS 7.7 vs 4.0 months; P=0.01) (N Engl J Med 2017;376:836−847). Here we aimed to identify baseline biomarkers that were prognostic (disease-related) or predictive (treatment-related) of clinical outcomes.Methods: Adults with R/R ALL (refractory to primary induction therapy or salvage therapy, first relapse <1 year, second or later relapse, or relapse after allogeneic hematopoietic cell transplantation [alloHCT]) were randomized to receive either blinatumomab or SOC. Blinatumomab was given by continuous infusion (4 wks on/2 wks off) for up to 5 cycles (9 μg/d on d1‒7 of cycle 1 and 28 μg/d thereafter). Up to 4 additional maintenance cycles (4 wks on/8 wks off) were allowed for up to 1 year. Evaluated biomarkers included leukocytes, monocytes, lymphocytes, granulocytes, platelets, neutrophils, CD45+ CD3+ CD4+ T cells, CD45+ CD3+ CD8+ T cells, CD45+ CD3- CD19+ B cells, CD3- CD16+ CD56+ cells, bone marrow (BM) blasts, CD3+ T cells, CD19+ cells, and ratios of CD3+/CD19+ and CD3+%/BM blast%. A multivariate model was used to select potential biomarkers associated with treatment outcomes. Effect on outcome was determined if either the main effect or interaction term between the biomarker and treatment had a P value ≤0.3. Prognostic and predictive associations of baseline biomarkers with OS, hematologic response (complete remission [CR] or CR with full, partial, or incomplete hematologic recovery [CR/CRh/CRi]) within the first 12 wks, and minimal residual disease (MRD) response were assessed by Cox regression for OS and multivariate logistic regression for hematologic response and MRD response. Predictive biomarkers were identified using interaction tests with treatment group.Results: A total of 405 patients received blinatumomab (n=271) or SOC (n=134). Baseline characteristics including age, prior alloHCT, salvage status, granulocytes, platelets, and lymphocyte subpopulations (CD45+ CD3+ CD8+, CD45+ CD3- CD19+, and CD3+) were generally balanced between treatment arms. Associations of baseline biomarkers with clinical outcomes are presented in the table. Higher platelet counts were associated with decreased risk of death (HR 0.43; 95% CI 0.29-0.65) and increased CR (odds ratio [OR] 4.12; 95% CI 2.04-8.33) and CR/CRh/CRi (HR 1.64; 95% CI 0.90-2.99). Higher granulocyte counts were associated with decreased risk of death (HR 0.75; 95% CI 0.57-0.99), whereas total leukocyte counts were not (HR 1.04; 95% CI 0.70-1.54). Lower BM blast percentage was associated with increased CR (OR 0.90; 95% CI 0.82-0.97) and CR/CRh/CRi (OR 0.87; 95% CI 0.80-0.95) but not OS (HR 1.04; 95% CI 0.98-1.09). Higher CD3+ T cells were associated with increased MRD response (OR 1.75; 95% CI 0.99-3.08). Higher counts of CD45+ CD3- CD19+ cells increased the risk of death for blinatumomab patients (HR 1.19; 95% CI 1.09-1.29) but also was not associated with a survival advantage for SOC patients (HR 1.03; 95% CI 0.93-1.15). Higher CD45+ CD3+ CD8+ T cells were associated with increased CR (OR 1.39; 95% CI 1.18-1.65) and CR/CRh/CRi (OR 1.44; 95% CI 1.22-1.70) in patients treated with blinatumomab but did not correlate with increased hematologic response in SOC patients. Multiple logistic regression modeling for adverse events (AEs) of interest showed that BM blasts and lymphocyte markers (CD3+ and CD45+ CD3+ CD8+) were not associated with grade ≥3 neurologic events, infections, or cytokine release syndrome.Conclusions: Higher platelet and granulocyte counts, lower BM blasts, and higher numbers of CD3+ T cells were prognostic for improved OS, hematologic response, and MRD response, respectively, in patients with R/R ALL who received blinatumomab vs SOC. A higher frequency of CD45+ CD3+ CD8+ cells was predictive of increased hematologic response to blinatumomab, whereas a higher frequency of CD45+ CD3- CD19+ cells was predictive of decreased OS. BM blast percentage and lymphocyte markers were not associated with AEs of interest. Overall, these data demonstrate that responses to blinatumomab can be seen across a variety of subgroups, including those expressing specific baseline biomarkers. Further study is needed to identify subgroups most likely to benefit from blinatumomab. [Display omitted] DisclosuresWei:AbbVie, Celgene, Servier: Research Funding; AbbVie, Celgene, Novartis, Amgen, Servier: Membership on an entity's Board of Directors or advisory committees; AbbVie, Celgene, Novartis, Amgen, Servier: Honoraria. Ribera:Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Pfizer: Research Funding, Speakers Bureau; Amgen Inc.: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; ARIAD: Research Funding, Speakers Bureau; Roche: Honoraria. Larson:Daiichi Sankyo: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen Inc.: Research Funding. Ritchie:Amgen Inc.: Honoraria. Chen:Amgen Inc.: Employment, Other: Stock holder. Dos Santos:Amgen Inc.: Employment. Zimmerman:Amgen Inc.: Employment, Equity Ownership. Kantarjian:Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding.

Comparison of Mobilized Peripheral Blood Stem Cells Versus Unmanipulated Bone Marrow Haploidentical Transplantation Using Post-Transplant Cyclophosphamide: A Retrospective Study of SFGM-TC in 176 Patients

IntroductionThe administration of high dose cyclophosphamide post-transplant (HD-Cy) has improved the outcome of patients who receive an unmanipulated haploidentical graft. The modalities described by the group of Baltimore originally associated a non myeloablative conditioning regimen and the use of a T cell replete bone marrow (BM) graft. Nevertheless, mobilized peripheral blood (PB) is increasingly used as it is more convenient. A very recent report from the United States showed a lower rate of graft versus host disease (GVHD) with BM but an increased risk of relapse in leukemic patients, as compared to PB, leading to a similar overall survival (OS). We report the French experience of haploidentical transplantation using HD-Cy and unmanipulated BM or PB, with a particular interest on the contents of the grafts.Patients and methodsOne hundred seventy-six patients with hematologic malignancy received a haploidentical transplant in 21 French centers from May 2012 to December 2015. We compared the outcome of patients who received a BM graft (n=89) with those receiving a PB graft (N=87). Median age at transplant was 44 year-old (2.5-72). One hundred thirteen (64%) patients had myeloid disease, 19 (11%) had acute lymphoblastic leukemia and 44 (25%) had lymphoid neoplasm. Prophylaxis of GVHD consisted in anticalcineurin and mycophenolate in 94% of the cases. All patients received HD-CY post-transplant (100 mg per kg total dose).ResultsThe 2 groups were comparable for age, disease status at transplant, type of conditioning regimen (RIC 71% and 80% for BM and PB respectively; p=0.16), recipient cytomegalovirus (CMV) seropositive status, ABO mismatch. PB recipients were more likely to receive antithymocyte globulin (ATG) (N=19 vs 0) and to have a higher disease risk index (DRI). The number of CD34+ and CD3+ infused cells was higher in PB grafts (median 2.64x106 CD34/Kg and 26 x106 CD3/Kg for BM and 6.03x106 CD34/Kg and 191 x106 CD3/Kg for PB. Median follow up was 281 days (6-1851).In univariate analysis, the rate of patients who recovered neutrophils and platelet (>50G/L) were similar in both groups (91% and 75% vs 96% and 75% for BM vs PB respectively). However, median time to neutrophil engraftment was significantly longer in BM than in PB group (20 days (13-59) vs 18 days (13-45) respectively, p=0.004). Median time to platelet recovery was not significantly different (30 days (17-250) vs 24 days (13-52) in BM and PB, p=0.14). Patients who recovered neutrophils received a graft which contained more CD3+ cells than patients who did not (p=0.007).At one year, OS, progression free survival (PFS) and relapse were similar in the 2 groups (OS = 64% vs 53% (p=0.1), PFS= 53% vs 46 % (p=0.17) and relapse rate 28% vs 34% (p=0.19) in BM and PB respectively). Acute GVHD II-IV or III-IV and chronic GVHD were similar in both groups (aGVHD II-IV= 18% vs 28% (p=0.09) and aGVHD III-IV = 7% vs 9% (p=0.48), cGVHD=13% vs 15% (p=0.6) in BM and PB respectively), resulting in a similar GVHD-free relapse-free survival (GRFS) at 1 year (48% vs 37% (p=0.09)). Interestingly, the number of infused CD3+ cells/Kg impacted the incidence of acute GVHD II-IV (p=0.049).High DRI and the lack of complete remission at transplant had a significant pejorative effect on OS, PFS, relapse, GRFS and platelet recovery. CMV seropositive recipients had a worse GRFS (p=0.045).ConclusionIn this retrospective study the use of PB instead of BM does not significantly impact the outcome of patients who received a haploidentical unmanipulated graft for a malignant disease with HD-CY. PB graft contained more CD3+ and CD34+ cells than BM and were associated with a faster neutrophils recovery. A higher number of CD3+ cells increased the risk of acute GVHD II-IV in univariate analysis, without significant effect on GRFS. Prospective randomized studies are needed to evaluate the effect of PB vs BM in haploidentical settings. DisclosuresPeffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Defining a Bone Marrow Immune Signature Associated with Immunoglobulin Isotype Switching after Autologous Stem Cell Transplant

Triplet induction therapy followed by autologous stem cell transplant (ASCT) and maintenance therapy can render long-term remission for newly diagnosed multiple myeloma (MM) patients (pts). Robust post-ASCT immune-reconstitution correlates with lower minimal residual disease (MRD) and better clinical outcomes but immune signatures to define such responses are lacking (Ho et al. Blood Advances 1.15 (2017). Following ASCT, the Immunoglobulin Isotype Switch (IIS) results in a new serum M-protein on serum protein electrophoresis (SPE), distinct from the M-protein pattern present at diagnosis, may be associated with a good prognosis. The objective of this study was to analyze humoral immune reconstitution that presents as IIS after ASCT and to determine if there was a correlation with patient or disease characteristics, transplant parameters and to define the associated immune signature.MethodsThe Institutional Review Board-approved study evaluated MM pts who underwent ASCT between 2007 and 2016. Data collection included patients' characteristics, induction therapy, mobilization regimen, graft characteristics including CD34 cell dose, myeloid and erythroid progenitor cells (multi-potential colony forming unit or CFU-GM and erythroid burst-forming unit (BFU-E). Percentage of NK cells (CD56(+) CD3(-) and CD16(+)CD3(-)), T-cells (CD3(+),CD56 (-)), B-cells (CD19(+)),and T-cell subpopulations (CD4(+), CD8(+),were measured with flow cytometry in pre- and post-ASCT bone marrow samples. IIS was defined as the appearance of a new serum monoclonal spike on SPE and immunofixation different from original heavy or light chain detected at diagnosis. Monoclonal proteins and quantitative immunoglobulins were assessed day+100 and with frequency of 2-8 times per year afterward.The start date for all time dependent variables was the day of transplant. The Kaplan-Meier method was used to assess overall survival (OS) and progression-free survival (PFS). Two-tailed log-rank tests compared OS and PFS curves. Factors with a P -value <0.2 in univariate analysis were included in multivariate analysis, which was performed using the Cox regression hazard model. All analyses were performed utilizing SAS 9.2 (SAS Institute, Cary, NC, USA).ResultsThe analysis included 177 patients with median age of 60.7 years. IIS was detected in 39 pts (22.0%) with a significantly higher incidence in pts who received lenalidomide before ASCT (11.2% vs. 30.4%, P=0.001). Post-ASCT IIS was more frequent in IgA, light chain disease, and in pts who experience very good partial and complete response post-ASCT rather than less optimal response (Table-1 and 2). The occurrence of IIS did not correlate with ASCT toxicity, i.e., mucositis duration or grade as well as length of hospital admission (Table 2). IIS occurred more frequently in pts without suppressed un-involved immunoglobulin (92% vs 8%, p=0.001).The presence of post-ASCT IIS correlated significantly with PFS and OS (Figure.1.A and B). Age, cytogenetics, response category, presence of IIS and low LDH correlated with PFS by univariate analysis. Cytogenetics, LDH and IIS presence were also significantly associated with response as determined by multivariate analysis. The median time between pre- and post-ASCT BM was 92 days (range 79-123). Pts with IIS showed a trend toward faster B cell recovery in comparison to pts without IIS, however this difference did not reach statistical significance. All pts had low CD4/CD8 ratio, due to low number of CD4 and higher number of CD8. Appearance of post-ASCT IIS was associated with statistically lower CD8 T cell percentages and higher CD4/CD8 ratio in comparison to pts without IIS (Figure.1.C), suggestive of faster immune reconstitution in this group of pts. All pts who relapsed had an isotype different from IIS, highlighting the benign nature of this phenomenon.ConclusionOur data are consistent with the benign nature of IIS that may emerge in certain patients following ASCT. Association of a robust recovery of specific immune cell subpopulations with IIS occurrence and superior survival outcomes may represent an anti-myeloma protective effect resulted from cellular and humoral immunoreconstitution. Efforts to better understand how specific immune subpopulations trigger and regulate the immune response to tumors may improve long-term control of MM. Further research to define underlying anti-tumor effect of ISS is needed. [Display omitted] DisclosuresDe Lima:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding. Malek:Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Respiratory Syncytial Virus Infection Induces Expression of Inducible Nitric Oxide Synthase, CD3, and CD8 in Naturally Occurring Pneumonia in Lambs

Abstract Respiratory syncytial virus (RSV) is an RNA virus that belongs to the Pneumovirus genus of the Paramyxoviridae family. The aim of this study was to evaluate the expressions of inducible nitric oxide synthetase (iNOS), CD3 (pan T cells), and CD8 (cytotoxic T cells) in lamb lungs naturally infected with RSV using immunohistochemistry (IHC). For this purpose, 100 pneumonic and 10 control lung tissue samples were taken from lambs slaughtered in the slaughterhouse after macroscopic examination. The streptavidin– peroxidase method (ABC) was used for IHC staining, and it revealed RSV positivity in 18 of 100 examined lungs with pneumonia (18%). These positive cases were then immunostained for iNOS, CD3, and CD8, and compared to controls. In all these cases, an increase in iNOS expression (100%) was detected, the higher number of CD3+ T lymphocytes was detected in 14 (78%) cases while CD8+ T lymphocytes were detected in five (28%) cases, only. Given the increase of iNOS immunoexpression in all RSV-positive cases and increase in the number of CD3+ T lymphocytes in most cases, it was concluded that iNOS and CD3+ T lymphocytes play an important role in the immune response in lamb pneumonia with naturally occurring RSV infection. With this study, the role of the mentioned markers was evaluated for the first time in lambs naturally infected with RSV.

The contribution of the immune response to enhanced colibacillosis upon preceding viral respiratory infection in broiler chicken in a dual infection model

Colibacillosis in chickens caused by avian pathogenic Escherichia coli (APEC) is known to be aggravated by preceding infections with infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). The mechanism behind these virus-induced predispositions for secondary bacterial infections is poorly understood. Here we set out to investigate the immunopathogenesis of enhanced respiratory colibacillosis after preceding infections with these three viruses. Broilers were inoculated intratracheally with APEC six days after oculonasal and intratracheal inoculation with IBV, NDV, aMPV or buffered saline. After euthanasia at 1 and 8 days post infection (dpi) with APEC, birds were macroscopically examined and tissue samples were taken from the trachea, lungs and air sacs. In none of the groups differences in body weight were observed during the course of infection. Macroscopic lesion scoring revealed most severe tissue changes after NDV-APEC and IBV-APEC infection. Histologically, persistent tracheitis was detected in all virus-APEC groups, but not after APEC-only infection. In the lungs, mostly APEC-associated transient pneumonia was observed. Severe and persistent airsacculitis was present after NDV-APEC and IBV-APEC infection. Bacterial antigen was detected by immunohistochemistry only at 1 dpi APEC, predominantly in NDV-APEC- and IBV-APEC-infected lungs. Higher numbers of CD4+ and CD8+ lymphocytes persisted over time in NDV-APEC- and IBV-APEC-infected tracheas, as did CD4+ lymphocytes in NBV-APEC- and IBV-APEC-infected air sacs. KUL01+ cells, which include monocytes and macrophages, and TCRγδ+ lymphocytes were observed mostly in lung tissue in all infected groups with transient higher numbers of KUL01+ cells over time and higher numbers of TCRγδ+ lymphocytes mainly at 8 dpi. qPCR analysis revealed mostly trends of transient higher levels of IL-6 and IFNγ mRNA in lung tissue after IBV-APEC and also NDV-APEC infection and persistent higher levels of IL-6 mRNA after aMPV-APEC infection. In spleens, transient higher levels of IL-17 mRNA and more persistent higher levels of IL-6 mRNA were observed after all co-infections. No changes in IL-10 mRNA expression were seen. These results demonstrate a major impact of dual infections with respiratory viruses and APEC, compared to a single infection with APEC, on the chicken respiratory tract and suggest that immunopathogenesis contributes to lesion persistence.

Enhancement of immune responses by co-stimulation of TLR3 - TLR7 agonists as a potential therapeutics against rabies in mouse model

Rabies is always fatal, when post-exposure prophylaxis is administered after the onset of clinical symptoms. To date, there is no effective treatment of rabies once clinical symptoms has initiated. Therefore, we aimed to provide evidences which indicate the promising effects of combination treatment with TLR agonists following rabies infection.Four groups of rabies infected-mice (10-mice/group) were treated with PolyI:C 50 μg (a TLR3 agonist), Imiquimod50 μg (a TLR7 agonist), (Poly + Imi)25 μg and (Poly + Imi)50 μg respectively.The immune responses in each experimental groups were investigated in the brain through evaluation of GFAP, MAP2, CD4, HSP70, TLR3, TLR7 and apoptotic cell expression as well as determination of IFN-γ, TNF-α and IL-4, levels. The treatment with combination of agonists (Poly + Imi)50 μg/mouse resulted a 75% decrease of mortality rate and better extended survival time following street rabies virus infection. Higher number of CD4+T cells, TLR3 and TLR7 expression in the brain parenchyma observed in the groups receiving both combined agonist therapies at the levels of 25 μg and 50 μg. In spite of decreased number of neuronal cell, significant higher number of astrocytes was shown in the group given (Poly + Imi)25 μg. The obtained results also pointed to the dramatic decrease of HSP70 expression in all groups of infected mice whereas higher number of apoptotic cells and Caspase 8 expression were recorded in (Poly + Imi)25 μg treated group. Furthermore, the cytokine profile consisting the increased levels of TNF-α, IFN-γ and IL-4 revealed that both humoral and cellular responses were highly modulated in combination therapy of 50 μg of Imiquimod and Poly I:C. Reduced viral load as quantified by real-time PCR of rabies N gene expression in the brain also correlated with the better survival of agonist-treated groups of mice. Based on obtained results, we have presented evidences of beneficial utilization of combined agonist therapy composed of TLR3/TLR7 ligands. This treatment regimen extended survival of infected mice and decreased significantly their mortality rate. We believe that the results of synergy-inducing protection of both TLR3/TLR7 agonists lead to the enhancement of innate immune responses cells residing in the CNS which warrant the studies to further understanding of crosstalk mechanisms in cellular immunity against rabies in the future.

Adjuvant nivolumab for hepatocellular carcinoma (HCC) after surgical resection (SR) or radiofrequency ablation (RFA) (NIVOLVE): A phase 2 prospective multicenter single-arm trial and exploratory biomarker analysis.

4070 Background: The NIVOLVE trial was designed to assess the efficacy and safety of nivolumab as an adjuvant therapy for HCC, and to identify biomarkers predictive of recurrence in patients after SR or RFA (Registration # UMIN 000026648). Methods: The trial involved 11 sites and was conducted in patients with HCC who showed a complete response after SR (n = 33) or RFA (n = 22) (ITT). Overall, 53 of 55 patients with Child-Pugh A received nivolumab (240 mg/body every 2 weeks (8 cycles)), followed by nivolumab (480 mg/body every 4 weeks (8 cycles)) within 6 weeks after SR or RFA. The primary endpoint was the 1 year recurrence-free survival rate (RFSR). The key secondary endpoint was RFS. Exploratory biomarker analysis included mutations, copy number alterations, and tumor mutation burden in tumor tissues. Techniques included next generation sequencing, immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded tissues (n = 31, 13 with recurrence and 18 without) for CD8, PD-1, PD-L1, Foxp3, and β-catenin, and ctDNA analysis using pre-nivolumab whole blood by deep sequencing (CAPP-seq; Avenio). Results: The 1-year RFSR and median RFS were 76.7% and 26.0 months (95% confidence interval (CI), 23.9–28.1), respectively, with no difference between SR and RFA. Copy number gains (CNGs) in WNT/β-catenin related genes ( APC, CTNNB1, TCF7L1, TCF7L2) (n = 8) correlated with shorter RFS (positive: 11.8 months vs. negative: not reached [NR]; p = 0.0003). IHC revealed that negative staining for PD-1 (p &lt; 0.0001), a low combined positive score for PD-L1 (p = 0.0113), a low number of CD8+ tumor infiltrating lymphocytes (TILs) (p = 0.0130), and positivity for Foxp3+ cells (p = 0.0076) correlated with recurrence. Treatment-related adverse events (AEs) (n = 53) were as follows: all grades, 68%; grades 3–4 (18.9%); and immune related AEs, 25%. HCC cases with low numbers of CD8+ TILs or cases positive for Foxp3+ cells (n = 16) showed a significantly shorter RFS (16.8 months [95% CI, 8.7–25.1]) than those with high numbers of CD8+ TILs and those positive for PD-1/PD-L1 expression (n = 15) (NR [95% CI,26.2months–NR]) (p &lt; 0.0001). HCC cases with activation of the WNT/β-catenin pathway assessed by IHC (n = 9) showed shorter RFS (17.0 months [95% CI,1.1–26.2]) than those without activation (n = 22) (NR [95% CI,24.7 months–NR]) (p = 0.0191). Patients positive for ctDNA (n = 10) before nivolumab tended to have shorter RFS than those without ctDNA (n = 30) (26.2 vs. NR). There was no correlation between TMB and RFS. Conclusions: The 1 year RFSR and RFS in the NIVOLVE trial were 76.6% and 26.0 months, respectively. No new safety signal was observed. CNG in WNT/β-catenin-related genes, activation of the WNT/β-catenin pathway, the presence of Foxp3+ cells, and low numbers of CD8+ TILs may predict recurrence after SR or RFA with adjuvant nivolumab. Clinical trial information: 000026648.

Immune contexture in high-risk soft tissue sarcomas (STS): A planned analysis of the ISG-STS-1001 randomized trial.

11572 Background: The characteristics of immune contexture and its prognostic and predictive value in STS is left to be understood. This planned analysis of the ISG-STS-1001 trial, which compared neoadjuvant anthracycline + ifosfamide (AI) vs a histology-tailored (HT) chemotherapy (ChT), was aimed at characterizing the immune contexture after neoadjuvant ChT and investigating any association with the risk of recurrence. Methods: Patients registered in the ISG-STS-1001 study (ID: NCT01710176) were included if they had tumor tissue available for Tissue MicroArray (TMA), which was performed in the area of the surgical specimen with the highest lymphocyte infiltrate. The following markers were analyzed with IHC and measured quantitatively: CD3, CD8, PD1, GranzymeB, Foxp3, CD20, CD163, and PDL1. The T-Distributed Stochastic Neighboring Entities (t-SNE) analysis was used to account for the co-expression of IHC markers in each tumor. The prognostic value of each marker for disease-free survival (DFS) was assessed. Results: This analysis was conducted in 256 of 435 study patients. AI and HT neoadjuvant ChT did not result in any different distribution of immune contexture. Conversely, differences were observed between ‘complex’ karyotype STS (ck-STS: LMS, MPNST, UPS, MFS, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma) and ‘simple’ karyotype STS (sk-STS: MLPS and SS). Ck-STS were enriched in both CD3+ and CD8+ cells compared to sk-STS. These cells displayed an heterogeneous distribution and were dispersed inside the tumor nest, keeping direct contact with sarcoma cells. Ck-STS also displayed an enrichment in Granzyme B+, and CD163+ cells. PDL1+ cells were occasionally identified and were more frequent in ck-STS, suggesting an immune-related expression. Most STS were negative for CD20+ cells, however, when present these cells were highly represented and organized in tertiary lymphoid-like structure. The t-SNE generated plot clustered tumors, the ‘cold’ mainly including sk-STS and the ‘hot’ mainly composed by ck-STS. In the ‘hot’ group, a cluster of tumors displayed an immune infiltrate enriched with a high number of CD3, CD8, GranzymeB, PD-1, and PDL-1+ cells. When the prognostic value of the immune markers was investigated, the presence of CD20+ cells was the only independent prognostic factor for DFS (HR=0.68, 95%CI 0.52-0.91) in a histology-stratified estimate adjusting for tumor size in cm (HR=1.07, 95%CI 1.03-1.12) and patient age (HR=1.0, 95%CI 0.97-1.02). Conclusions: Immune contexture differed across sarcoma histologies after neoadjuvant ChT, rather than across the two study arms, with ck-STS being marked by a rich immune contexture. While a CD20+ infiltrate was found to be an independent prognostic factor for a better outcome, further analyses are in progress on the prognosis of patients with the richest immune contexture. Clinical trial information: NCT01710176.

Clinicopathologic, genomic, and tumor microenvironment correlates of aneuploidy and immunotherapy outcomes in NSCLC.

9119 Background: Cancer aneuploidy, an unbalanced number of chromosomes, is associated with somatic mutation rate, expression of proliferative genes, and altered immune signaling. Whether aneuploidy correlates to a distinct immunophenotype or impacts clinical outcomes to immune checkpoint inhibitors (ICIs) in NSCLC is unclear. Methods: In NSCLCs which underwent targeted next-generation sequencing, we retrospectively analyzed the aneuploidy score (AS), defined as the sum of the number of altered chromosome arms. An unbiased recursive partitioning (URP) algorithm was used to investigate an AS cutoff to discriminate responders from non-responders to ICIs. Multiplexed immunofluorescence to quantify CD8+, Foxp3+, PD-1+, and PD-L1 expression was performed to determine differences in tumor immune cells subsets according to AS cutoff. Results: Among 436 NSCLCs identified, stage I tumors (median AS 1) had significantly lower median AS (mAS) than stage IV cancers (mAS 7, P &lt; 0.001), stage III (mAS 4, P = 0.03), and numerically lower compared to stage II cancers (mAS 3, P = 0.18). We found no difference in the mAS across tumors with a PD-L1 tumor proportion score of ≥50%, 1-49%, or &lt; 1% (mAS 5 vs 7 vs 6, respectively, P = 0.26), nor was there any correlation between aneuploidy and TMB when taken as continuous variables (Spearman R: 0.074, P = 0.12). A total of 279 advanced NSCLCs with available aneuploidy scores were treated with ICIs. An URP analysis identified an AS of 2 as the strongest discriminator of objective response to ICI. Compared to pts with an AS &gt; 2 (N = 207, 74.2%), pts with AS ≤2 (N = 72, 25.8%) had a significantly higher objective response rate (ORR 43.0% vs 19.8%, P &lt; 0.001), a significantly longer median progression-free survival (mPFS 6.2 vs 2.9 months, HR: 0.70 [95% CI: 0.52-0.94], P = 0.02), and a significantly longer median overall survival (mOS 19.8 vs 13.8 months, HR: 0.66 [95% CI: 0.47-0.94], P = 0.02) to treatment with ICIs. After adjusting for other variables such as performance status, presence of oncogenic driver mutation, PD-L1, TMB, and line of treatment, AS was significantly associated with improved mPFS (HR: 0.72 [95% CI: 0.52-0.99], P = 0.04) and mOS (HR: 0.64 [95% CI: 0.44-0.94], P = 0.02). By contrast, among pts who received first-line platinum doublet chemotherapy without ICI, an AS ≤2 (N = 29), when compared to an AS &gt; 2 (N = 56), did not correlate with improved ORR (55.2% vs 44.6%, P = 0.4) or PFS (5.3 vs 4.8 months, HR 0.83 [95% CI: 0.5-1.3], P = 0.43). Among 179 NSCLCs profiled by multiplex immunofluorescence, compared to cancers with an AS &gt; 2, those with low aneuploidy had significantly higher numbers of CD8+, Foxp3+, PD-1+ immune cells, and PD-1+ CD8+ T cell, both intratumorally and when looking at the total numbers of cells within the tumor and at the tumor-stroma interface. Conclusions: NSCLCs with low aneuploidy have a distinct immune microenvironment and more favorable outcomes to ICIs.

Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.

High Dose Intravenous Vitamin C for Preventing The Disease Aggravation of Moderate COVID-19 Pneumonia. A Retrospective Propensity Matched Before-After Study.

Background: Coronavirus disease 2019 (COVID-19) pandemic is continuing to impact multiple countries worldwide and effective treatment options are still being developed. In this study, we investigate the potential of high-dose intravenous vitamin C (HDIVC) in the prevention of moderate COVID-19 disease aggravation. Methods: In this retrospective before-after case-matched clinical study, we compare the outcome and clinical courses of patients with moderate COVID-19 patients who were treated with an HDIVC protocol (intravenous injection of vitamin C, 100 mg/kg/day, 1 g/h, for 7 days from admission) during a one-month period (between March 18 and april 18, 2020, HDIVC group) with a control group treated without the HDIVC protocol during the preceding two months (January 18 to March 18, 2020). Patients in the two groups were matched in a 1:1 ratio according to age and gender. Results: The HDIVC and control groups each comprised 55 patients. For the primary outcomes, there was a significant difference in the number of patients that evolved from moderate to severe type between the two groups (HDIVC: 4/55 vs. control: 12/55, relative risk [RR] = 0.28 [0.08, 0.93], P = 0.03). Compared to the control group, there was a shorter duration of systemic inflammatory response syndrome (SIRS) (P = 0.0004) during the first week and lower SIRS occurrence (2/21 vs 10/22, P = 0.0086) on Day 7 (6–7 days after admission). In addition, HDIVC group had lower C-reactive protein levels (P = 0.005) and higher number of CD4+ T cells from Day 0 (on admission) to Day 7 (P = 0.04).” The levels of coagulation indicators, including activated partial thromboplastin time and D-dimer were also improved in the HDIVC compared to the control group on Day 7. Conclusion: HDIVC may be beneficial in limiting disease aggravation in the early stage of COVID-19 pneumonia, which may be related to its improvements on the inflammatory response, immune function and coagulation function. Further randomized controlled trials are required to augment these findings.

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation.

Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation. Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment. High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH. We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.

Clinical significance of CD8+ and FoxP3+ tumor-infiltrating lymphocytes and MFG-E8 expression in lower rectal cancer with preoperative chemoradiotherapy.

Preoperative chemoradiotherapy (CRT) for rectal cancer contributes to tumor down-staging and decreases locoregional recurrence. However, each patient shows a significantly different response to CRT. Therefore, the identification of predictive factors to CRT response would be beneficial to avoid unnecessary treatment. Cancer immunity in patients has been suggested to play an important role in the eradication of the tumor by CRT. In the present study, the utility of CD8+ and forkhead box P3 (FoxP3)+ tumor-infiltrating lymphocytes (TILs) and the expression of a novel immuno-regulatory factor, lactadherin (MFG-E8), in predicting CRT effectiveness in patients with rectal cancer was examined. A total of 61 patients with rectal cancer, who underwent curative resection following CRT were included in the study. The numbers of CD8+ and FoxP3+ TILs in a biopsy taken before CRT and MFG-E8 expression level in the specimens obtained at the time of the surgery after CRT were examined using immunohistochemical staining, and their association with clinicopathological characteristics, including patient survival, was determined. The tumors with more CD8+ TILs in the biopsy samples before CRT showed a significantly more favorable CRT response. The patients with tumors and a higher number of CD8+ TILs before CRT also exhibited significantly longer disease-free and overall survival times. Higher MFG-E8 expression level in post-CRT specimens was significantly associated with favorable CRT response; however, no significant association was found with any other clinicopathological characteristics, including survival time. The number of CD8+ TILs before CRT was a valuable predictor for CRT response and was associated with favorable prognosis in patients with lower rectal cancer and who were treated with CRT. High MFG-E8 expression level after CRT was also associated with a favorable CRT response.

Immune classification of clear cell renal cell carcinoma

Since the outcome of treatments, particularly immunotherapeutic interventions, depends on the tumor immune micro-environment (TIM), several experimental and computational tools such as flow cytometry, immunohistochemistry, and digital cytometry have been developed and utilized to classify TIM variations. In this project, we identify immune pattern of clear cell renal cell carcinomas (ccRCC) by estimating the percentage of each immune cell type in 526 renal tumors using the new powerful technique of digital cytometry. The results, which are in agreement with the results of a large-scale mass cytometry analysis, show that the most frequent immune cell types in ccRCC tumors are CD8+ T-cells, macrophages, and CD4+ T-cells. Saliently, unsupervised clustering of ccRCC primary tumors based on their relative number of immune cells indicates the existence of four distinct groups of ccRCC tumors. Tumors in the first group consist of approximately the same numbers of macrophages and CD8+ T-cells and and a slightly smaller number of CD4+ T cells than CD8+ T cells, while tumors in the second group have a significantly high number of macrophages compared to any other immune cell type (P-value <0.01). The third group of ccRCC tumors have a significantly higher number of CD8+ T-cells than any other immune cell type (P-value <0.01), while tumors in the group 4 have approximately the same numbers of macrophages and CD4+ T-cells and a significantly smaller number of CD8+ T-cells than CD4+ T-cells (P-value <0.01). Moreover, there is a high positive correlation between the expression levels of IFNG and PDCD1 and the percentage of CD8+ T-cells, and higher stage and grade of tumors have a substantially higher percentage of CD8+ T-cells. Furthermore, the primary tumors of patients, who are tumor free at the last time of follow up, have a significantly higher percentage of mast cells (P-value <0.01) compared to the patients with tumors for all groups of tumors except group 3.

CD8+\u2009T cells in the central nervous system of mice with herpes simplex infection are highly activated and express high levels of CCR5 and CXCR3

Herpes simplex virus type 2 (HSV-2) is a neurotropic virus that can cause meningitis, an inflammation of the meninges in the central nervous system. T cells are key players in viral clearance, and these cells migrate from peripheral blood into the central nervous system upon infection. Several factors contribute to T cell migration, including the expression of chemokines in the inflamed tissue that attract T cells through their expression of chemokine receptors. Here we investigated CD8+ T cell profile in the spinal cord in a mouse model of herpes simplex virus type 2 neuroinflammation. Mice were infected with HSV-2 and sacrificed when showing signs of neuroinflammation. Cells and/or tissue from spinal cord, spleen, and blood were analyzed for expression of activation markers, chemokine receptors, and chemokines. High numbers of CD8+ T cells were present in the spinal cord following genital HSV-2-infection. CD8+ T cells were highly activated and HSV-2 glycoprotein B -specific effector cells, some of which showed signs of recent degranulation. They also expressed high levels of many chemokine receptors, in particular CCR2, CCR4, CCR5, and CXCR3. Investigating corresponding receptor ligands in spinal cord tissue revealed markedly increased expression of the cognate ligands CCL2, CCL5, CCL8, CCL12, and CXCL10. This study shows that during herpesvirus neuroinflammation anti-viral CD8+ T cells accumulate in the CNS. CD8+ T cells in the CNS also express chemotactic receptors cognate to the chemotactic gradients in the spinal cord. This indicates that anti-viral CD8+ T cells may migrate to infected areas in the spinal cord during herpesvirus neuroinflammation in response to chemotactic gradients.