Percentage Of T-cells Research Articles

BCL11B Mutations Are Associated with Higher CD8+ T-Cell Percentage and Favorable Clinical Outcomes in Patients with T-Cell Acute Lymphoblastic Leukemia.

BCL11B Mutations Are Associated with Higher CD8+ T-Cell Percentage and Favorable Clinical Outcomes in Patients with T-Cell Acute Lymphoblastic Leukemia.

442. Clinical Outcomes of Checkpoint Inhibitor Therapy in People with HIV: A Single-Center Case Series

Abstract Background Immunotherapeutics, particularly immune-checkpoint inhibitors (ICI), have transformed the treatment landscape for advanced cancer. Such therapies could considerably benefit people with HIV (PWH), yet PWH have often been excluded from ICI trials such that data regarding ICI safety in PWH are limited. In addition, ICIs have recently been proposed as latency reversing agents that could impact the HIV reservoir. Here, we report the outcomes in a cohort of PWH after ICI therapy. Methods Using the electronic health record, we systematically identified all PWH with advanced cancer who received ICIs from January 2000 to December 2018 at one academic medical center. We collected data on demographics, HIV history, and HIV and hematologic parameters before and after ICI in 25 PWH over 29 treatment episodes. We cataloged adverse events following treatment. Results In this cohort, ICI therapy was not associated with a significant increase in plasma HIV RNA levels nor with significant change in CD4+ T-cell count, CD8+ T-cell count, or CD4/CD8 ratio (Figure 1). Among 19 treatment episodes with available pre- and post- treatment data, there were five viral blips (26%); HIV RNA exceeded 200 copies/mL in one episode. The blip rate observed among PWH on ART in the literature ranges from 10-30% and among PWH receiving ICI from 0-33%. Adverse events included infectious and autoimmune conditions across multiple organ systems including systemic, pulmonary, gastrointestinal/hepatic, genitourinary, neurologic, endocrine, and dermatologic. The most common adverse events were diarrhea (n=7) and rash (n=5). Five deaths occurred within one month of ICI initiation; all were attributed to progressive malignancy. Figure 1:Changes in HIV parameters associated with checkpoint inhibitor therapy (a) Pretreatment plasma HIV RNA, n=23. There was one episode in which the pretreatment plasma HIV RNA was greater than 200 copies/ml. (b) Pretreatment CD4+ Tcell counts, n=24. (c) Longitudinal plasma HIV RNA trends in 5 individuals who experienced a viral blip, defined as an increase in plasma HIV RNA from “not detected” to “detected” or greater within a year of ICI initiation. Symbols represent unique participants. (d) Pretreatment and posttreatment plasma HIV RNA values closest to ICI initiation, n=19. Points are scaled to proportion of sample. (e) Pretreatment and posttreatment CD4+ T-cell counts closest to ICI initiation, n=20. Thick line represents the change in median M between the two time points. (f) Pretreatment and posttreatment CD4+ T-cell percentages closest to ICI initiation, n=19.Thick line represents the change in median M between the two time points. (g) Pretreatment and posttreatment CD8+ T-cell counts closest to ICI initiation, n=18. Thick line represents change in median M between the two time points. (h) Pretreatment and posttreatment CD4/CD8 ratios closest to ICI initiation, n=18. Thick line represents the change in median M between the two time points. Conclusion This single-center cohort demonstrated no significant impact on HIV parameters among PWH receiving ICI. This study contributes to the growing body of evidence suggesting the safety and tolerability of ICI in PWH in both HIV and non-HIV-related outcomes and supports the inclusion of PWH in future ICI clinical trials. While we did not see an increased rate of viral blips on routine clinical testing, our findings also provide key preliminary data supporting the safety and tolerability of ICIs in future studies of these agents as HIV therapeutics, for example as agents that may reverse HIV latency. Disclosures All Authors: No reported disclosures.

Long-term effects of ciclosporin and oclacitinib on mediators of tolerance, regulatory T-cells, IL-10 and TGF-β, in dogs with atopic dermatitis.

Atopic dogs often are managed with allergen-specific immunotherapy (AIT) and concurrent dosages of ciclosporin (CSA) or oclacitinib to alleviate their clinical signs. Both drugs might affect proper tolerance induction by inhibiting regulatory T-cell (Treg) induction. We evaluated Treg cell numbers and serum interleukin (IL)-10 and transforming growth factor-beta (TGF-β)1 levels in dogs diagnosed with atopic dermatitis (AD) and successfully treated with either CSA or oclacitinib for nine or more months. We included 15 dogs receiving oclacitinib, 14 dogs treated with CSA, 15 healthy dogs, 13 dogs with untreated moderate-to-severe AD and 15 atopic dogs controlled with AIT. Peripheral blood CD4+CD25+FOXP3+ T-cell percentages were determined using flow cytometry. Serum concentrations of IL-10 and TGF-β1 were measured by enzyme-linked immunosorbent assay. The percentage of Treg cells in the CSA group was significantly lower in comparison with the healthy group (p=0.0003), the nontreated AD group (p=0.0056) or the AIT group (p=0.0186). There was no significant difference in Treg cell percentages between the CSA and oclacitinib groups, nor between the oclacitinib and the healthy, nontreated AD or AIT-treated dogs. No significant differences were detected in IL-10 and TGF-β1 serum concentrations between the five groups. Lower Treg cell percentages in the CSA-treated dogs suggest an impact of this drug on this cell population; however, it does not necessarily mean that it diminishes tolerance. Functionality and cytokine production may be more important than the number of Treg cells. Further studies evaluating the treatment outcome of dogs receiving AIT and concurrent drugs are needed to show clinical relevance.

Regulatory T-Cell Enhancement, Expression of Adhesion Molecules, and Production of Anti-Inflammatory Factors Are Differentially Modulated by Spheroid-Cultured Mesenchymal Stem Cells.

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3D-MSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell-cell adhesion molecules for optimal MSC immunomodulatory function.

Biological Differences between Axi-Cel and Tisa-Cel Products and Impacts on Outcomes in Lymphoma Patients

Purpose Axicabtagene ciloleucel (axi-cel, Yescarta® (Axi)) and tisagenlecleucel (tisa-cel, Kymriah® (Tisa)) have been approved and commercialized in Europe for Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL) after 2 or more previous lines of therapy. A recent Lysa Study from a matched analysis of the Descar-T registry has reported that Axi may yield a higher disease control rate compared to Tisa but at the expense of a higher toxicity[Bachy et al. EHA 2022]. Biological reports have shed light on the importance of both expansion and persistence of CAR T-cells to enhance disease control. In addition, in a mouse model, viability levels of CAR T cells both immediately after thawing and after 1 day of in-vitro culture were reported to be important indicator of the CAR-T cells antitumor activity [Zah E et al. Can Im Res. 2016]. In this single-center prospective study, we analyzed biological characteristics of both Axi and Tisa anti-CD19 CAR T-cells infused in 48 patients and looked at their correlation with outcomes. Experimental design From March 2019 to June 2022, residual cells were obtained after washing 48 infusion product bags, 27 Axi and 21 Tisa products. Cells' viability was analyzed immediately, and percentage of CAR positive T-cells was determined by flow cytometry with CD19 CAR detection reagent (Miltenyi Biotec). Cells were put in complete medium (RPMI, glutamax, 8% of human serum and 300 UI of IL-2/ml) at 37°C, viability was analyzed after 1 days of in-vitro culture and doubling population number was followed for 1 to 4 weeks. Results Among the 48 patients (median age: 62 yo, range: 37-78), 56% had a DLBCL, and 29% had a transformed Follicular Lymphoma. Median number of previous lines was 3 (range, 2-10), which included an ASCT in 37.5% of patients. No differences in terms of characteristics were observed between Tisa and Axi cases. Tisa displayed a lesser cell viability (median 57%, range: 28-78) as compared to Axi (81%; range 62-94, p<0.001, (Figure 1). After 24H, the median percentage (%) of live cell recovery were similar (Tisa 89%, vs Axi 84%; p=0.23). However, 43% of Tisa and 20 % of Axi preparations displayed a viable cell yield ≤ 50% the day after thawing. The recommended doses of viable CAR+ T cells to be injected in patients is 0.6 to 6.0 x 108 cells for Tisa and 2 × 106/kg, with a maximum of 2 × 108 cells, for Axi. All patients received the recommended dose even if Tisa products were characterized by a significantly lower fraction of CD3+ CAR+ T-cells as compared to Axi (median 27% , range: 14-53 vs. 70%; range 37-88 P<0.001) (Figure 1). Median CD4/CD8 ratii among CAR+ T cells were similar between both products (Tisa: 3.5 vs Axi 2.3, p=0.35). In vitro and in the absence of antigen stimulation, total T-cells were able to proliferate, Tisa product had a higher amplification capacity (median nbr of division = 8.8 vs 3.1, p=. 6e-6). Finally, the proportion of CAR negative T cells (mostly activated T-cells) injected was highly variable between patients and ranged from 1.3.108 to 61.4.108 for Tisa while it was only from 0.2.108 to 3.4.108 for Axi. All but one patient infused with Axi presented with cytokine release syndrome (CRS) compared to 62% of patients infused with Tisa (p=0.006); the same significant difference was observed for immune effector cell-associated neurotoxicity syndrome (ICANs) (67% vs 14%, p<0.001). Conversely, survivals were comparable in this small cohort. Only the percentage of CD3+ CAR+ T-cells had a clinical impact. Indeed, a significant higher median % of these cells was found in patients developing all grade CRS (58% [range, 24-88%] vs 25% [range, 14-73%], p=0.014). (Figure 2) This was also true when considering only patients receiving Tisa (CRS+ 36.5 % [range, 24-88%] vs CRS- 24% [range, 14-73%], p=0.02. No biological association with ICANs nor hematological toxicity could be reliably reported in this cohort. Discussion In this study, four major biological differences were observed between Tisa and Axi products: the cell viability, the proliferative activity in vitro, the % of CAR positive T-cells and the total amount of CAR negative T-cells injected into the patient. However, impact on outcomes was modest as only CRS were found to be correlated to the percentage of CD3+ CAR+ T-cells injected, of note Axi-cell which was reported to be associated with a better disease control rate developed more CRS in our cohort, which is line with a high and consistent percentage of CD3+ CAR+ T-cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

8-Day Versus 12-Day Manufactured LV20.19 CAR T-Cells for B-Cell Non-Hodgkin Lymphoma

Introduction: CD19 CAR T-cell therapy has revolutionized outcomes for patients (pts) with relapsed, refractory (R/R) B-cell non-Hodgkin lymphoma (NHL). However, the vein-to-vein time for commercially approved CAR-T cell products and downregulation of the target receptor with CD19 CAR-T remain limitations of therapy. To improve clinical outcomes we utilized a lentiviral, bispecific, tandem anti-CD20/anti-CD19 (LV20.19) CAR T-cell in pts with R/R B-cell NHL with point of care manufacturing (MF) to limit time to infusion. For MF, we used IL-7+IL-15 for cell expansion with two arms comparing a flexible 8/12 day MF process versus (vs) a fixed 12 day platform. We hypothesized shorter MF would produce a more naïve CAR T-cell population improving clinical responses for R/R B-cell NHL. Methods: We conducted a Phase 1/2, multi-cohort, single center, prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Cells were manufactured via CliniMACS Prodigy device. Pts were sequentially enrolled on the two arms: fixed 12-day MF vs flexible 8/12 day MF where cells were harvested on day 8 if target dose was met. The goal was fresh infusion for all pts with lymphodepletion initiated during the MF process. After safety run-in, we opened two Phase 1b expansion cohorts. Descriptive statistics were utilized for baseline characteristics and Fischer's exact test for comparison of categorical variables. Survival analyses were calculated utilizing the Kaplan-Meier method and differences between groups were evaluated via log-rank test. Results: In total, there were 21 pts with DLBCL or FL treated with LV20.19 CAR-T cells with 11 pts treated on the flexible 8/12 MF arm and 10 pts treated on the fixed 12-day MF arm. MF success was 100% and 18 (86%) pts received fresh cells without cryopreservation. All 11 pts on the flexible 8/12 arm met goal target dose at Day 8 of MF. The median age of all pts was 66 years (41-78). Median prior lines of therapy were 3 (2-11). Nine (43%) pts progressed after prior autologous transplant and 2 pts progressed after anti-CD19 CAR T-cell therapy (Table 1). By cohort, there was no difference in day 28 overall response rates: 82% with an 8-day process vs 70% with a 12-day process (p=0.64). Both prior CAR treated pts progressed at day 28. Day 28 complete response (CR) rates trended higher in the 8-day arm (64% vs 20%, p=0.08). There was no difference in median duration of response (DOR); 6 months in the 8-day arm vs not reached in the 12-day arm (p=0.64). The 1-year OS for all pts was 61%. There were 4 non-relapse mortality events in responding pts beyond day 28 (1=therapy related MDS, 1=covid, 1=line associated sepsis, 1=pneumonia). More pts in the 8-day arm had CRS, 100% (n=11) with 1 grade 3 vs 70% (n=7) in the 12-day arm, all grade 1-2 (p=0.08). Immune effector cell associated neurotoxicity syndrome (ICANS) occurred in 2 pts in the 8-day arm, both grade 1-2 and in 4 pts in the 12-day arm (2=grade 2 and 2=grade 3). Immunophenotypically CAR T-cells harvested at day 8 had significantly higher numbers of central memory (CM) T-cells and lower terminally differentiated effector memory re-expressing CD45RA (EMRA) T-cells in both CD4 and CD8 compartments than CAR T-cells harvested at day 12 (Figure 1). Intra-patient immunophenotyping of 3 pts assigned to the 12-day MF arm demonstrated a similar shift with decreasing CM T-cells and increasing EMRA T-cells between day 7 (in-process testing) and day 12 (harvest). Conclusions: Bispecific LV20.19 CAR T-cells are safe and efficacious for pts with R/R B-cell NHL. Shorter MF produced a more naïve CAR T-cell product with no difference in toxicity profile. Higher percentage of CM T-cells and lower EMRA T-cells were observed earlier in the MF process both between the two cohorts and intra-patient. While CR rates were higher in the 8-day arm, duration of response was similar in both groups. These data confirm that a shorter MF process is feasible and improves immunophenotype of the final CAR product without impacting safety. Future studies will utilize an 8-day MF process. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The influence of stimulated thyroglobulin and lymphocyte subsets before radioiodine therapy on the therapeutic response in patients with intermediate- and high-risk papillary thyroid carcinoma.

The aim of the present study was to investigate the factors influencing the short-term response to the initial radioiodine therapy (RT) course in patients with intermediate- and high-risk papillary thyroid carcinoma (PTC). A total of 182 patients with intermediate- and high-risk PTC who underwent RT in our hospital from March 2018 to October 2020 were retrospectively enrolled. The patients were divided into incomplete response (IR) and nonincomplete response (Non-IR) groups according to the response observed in clinical follow-up within 6-12months after RT. Univariate and multivariate logistic regression analyses were used to investigate the effects of 15 observed factors on the response to RT. Receiver operating characteristic (ROC) curve analysis was used to determine the value of factors found to be significant in multivariate analyses for predicting an IR. A total of 182 patients with intermediate- and high-risk PTC were analyzed; the percentage of patients with a Non-IR was 61.54% (112/182), and the percentage of patients with an IR was 38.46% (70/182). The CD4+ T-cell percentage (t = 4.757, P = 0.000), CD4/CD8 (z = - 2.632, P = 0.008), stimulated thyroglobulin (sTg) level (z = - 8.273, P = 0.000) and M stage (χ2 = 17.823, P = 0.000) of the two groups were significantly different. Multivariate analysis showed that only the sTg level (OR: 1.116, 95% CI 1.068-1.165, P < 0.001) and CD4+ T-cell percentage (OR: 0.909, 95% CI 0.854-0.968, P = 0.003) were independent factors associated with the therapeutic response to RT. The cutoff sTg level and CD4+ T-cell percentage for predicting an IR were 7.62μg/L and 40.95%, respectively. The sTg level and CD4+ T-cell percentage were verified to be independent predictive factors of RT response. Higher sTg levels and lower CD4+ T-cell percentages were related to an IR in patients with intermediate- and high-risk PTC.

Lymphocyte Subsets in Cervicovaginal Lavage Specimens of HIV-Infected Women: A Surrogate Risk Marker of HPV-Associated Cervical Lesions

Introduction: To better understand the role of mucosa immunity in the development of cervical carcinoma in HIV infection, cervical lymphocyte subsets were characterized in HIV+ and HIV− women, as well as their relation to HPV-associated cervical lesions. Methods: Eighty-three (52 HIV+, 31 HIV−) cell suspensions of cervicovaginal lavage (CVL) and 52 HIV+ peripheral blood (PB) samples were assessed by flow cytometry to evaluate lymphoid populations. High-risk (HR) HPV was assessed in liquid-based cytology and HIV mRNA in PB in the same patients. Results: Cervical CD4⁺ T cells and CD4⁺/CD8⁺ ratio were decreased (p < 0.0001) and cervical CD8⁺ T cells were increased (p = 0.0080) in HIV+ women. These patients had lower CD4⁺ T-cell percentages in CVL compared to PB (p = 0.0257), and the opposite was true for CD8⁺ T cells (p = 0.0104). They also had a higher prevalence of high-grade squamous intraepithelial lesions (SILs) with an increased prevalence of HR HPV. Cervical CD8⁺ T cells were increased in HR HPV+ patients (p = 0.0300) and related to higher prevalence of SILs (p = 0.0001). Discussion/Conclusion: Cervical lymphoid populations can be characterized by flow cytometry, showing a distinct cervical T-cell compartment in HIV+ women. This may represent a surrogate risk marker of HPV-associated cervical lesions in this population and prompt further research on this subject, contributing to improving patients’ management.

Effects of a randomized-controlled trial of cognitive behavioral stress management: Psychosocial adaptation and immune status in men with early-stage prostate cancer

ObjectiveTargets of intervention in cognitive behavioral stress management (CBSM), such as benefit finding (BF) and perceived stress management skills (PSMS), may counteract stress-related changes that impact the immune system. This study tested whether BF, PSMS, and optimism influence the effects of CBSM on immune status in men with prostate cancer. MethodsMen with prostate cancer were randomized to receive CBSM or a psychoeducation (PE) control comparison (NCT05486754). Life Orientation Test-Revised assessed baseline optimism. The Benefit Finding Scale and Measure of Current Status measured BF and PSMS after CBSM. T-cells and T-helper cells captured immune status change at baseline and 6-months post-CBSM. MPlus and SPSS (PROCESS) tested condition effects and moderated mediation, controlling for covariates. Results256 primarily middle-aged, White Non-Hispanic or Hispanic men enrolled. PSMS mediated CBSM effects on T-cell and T-helper cell percentage, such that T-cell and T-helper cell percentages were reduced in men in CBSM versus PE via PSMS. Optimism moderated this mediation with the mediating effect of PSMS only observed among men with average optimism versus those with low or high optimism. ConclusionBaseline psychological characteristics, as well as limited specificity of immune measurement, could explain the conditional effects in this sample.Trial Registration: NCT05486754

P041: Low percentage of T lymphocytes in Hodgkin’s Lymphoma lymph nodes, measured by flow cytometry, is associated with inferior progression free survival regardless of negative interim pet scan status

Figure 1: Kaplan-Meier estimates of progression free survival stratified according to T-lymphocyte percentage in lymph node biopsies in Hodgkin lymphoma Background: Tumor microenvironment (TME) can influence antitumor immunity and survival in Hodgkin lymphoma (HL) as previously published. However, most of this data has sparse clinical applicability. Flow cytometry (FC) has limited utility in HL diagnosis but its potential to assess TME has not been fully explored yet. Aims: To determine the prognostic impact on survival of the percentage (%) of tumor infiltrating lymphocytes (TIL) and CD4/CD8 ratio in lymph nodes (LN) measured by FC. Methods: We selected patients (pts) with HL, available LN FC data at diagnosis and i-PET scan, treated at our center from 2012–2020. FC was performed with 8-color panels according to Euroflow protocols. TIL % and CD4/CD8 ratio in LN by FC were compared to normal values reported (Battaglia, Immunology 2003) and stratified as low, normal and high. Response was assessed per Lugano recommendations. Progression free survival (PFS) was estimated with Kaplan-Meier, multivariate analysis with Cox regression, and variables were compared with log-rank test. Results: 46 pts were included. Median age: 34 years. 24/27 with early stage had unfavorable risk and 11/19 with advanced disease had IPS≥3. 95% received ABVD. Complete response rate was 91.3%. 2 pts died. Median PFS: not reached (NR), 75th percentile 43 months (m), and median follow up: 49 m. i-PET negative (neg) pts had significantly better PFS (median NR vs 13.7 m in i-PET positive subgroup, p:0.014). Low LN TIL % was associated with inferior PFS (median 24.4 m vs. NR in pts with normal or high TIL, p:0.024). Regarding CD4/CD8 ratio, PFS rates were 100%, 75% and 60% in normal, low and high subgroups respectively, p:0.041. Both low TIL and positive i-PET remained independent predictors of survival on multivariate analysis with HR of 11.8 (CI 95% 1.9–72.7, p:0.021), and 7.7 (CI 95% 1.6–36.4, p:0.01) respectively. LN TIL % could further stratify outcomes in pts with neg i-PET scans. In this subset, pts with low TIL had a median PFS of 24.4 m and PFS rates of 33.3% vs. median NR in normal and high subgroups and PFS rates of 88.9 and 87.5% respectively, p:0.006. Conclusions: LN TIL % measured by FC showed prognostic impact in our cohort of HL pts. Pts with low TIL had inferior PFS, even in those with optimal interim response where the median was only 2 years. This finding denotes the critical role of T cells to achieve cure. Checkpoint blockade might constitute an appealing approach in these pts to restore effective antitumor immunity.

Sixteen-Week Vitamin D3 Supplementation Increases Peripheral T Cells in Overweight Black Individuals: Post hoc Analysis of a Randomized, Double-Blinded, Placebo-Controlled Trial.

Background: Vitamin D is considered to modulate T-cell function, which has been implicated in the treatment of inflammatory conditions. However, there is limited knowledge on the effects of vitamin D and its influences on circulating T-cell profiles in humans, particularly in overweight Black individuals who are more likely to be vitamin D insufficient (serum 25(OH)D concentrations of ≤20 ng/mL). Thus, this study tested the hypothesis that vitamin D supplementation modulates T-cell composition, which is in a dose-dependent manner. Methods: A 16-week randomized, double-blinded, placebo-controlled trial of vitamin D3 supplementation was undertaken in 70 overweight/obese Black people (mean age = 26 years, 82% female) with 25 hydroxyvitamin D ≤ 20 ng/mL at baseline. Subjects were randomly assigned a supervised monthly oral vitamin D3 equivalent to approximately 600 IU/day (n = 17), 2000 IU/day (n = 18), 4000 IU/day (n = 18), or a placebo (n = 17). Fresh peripheral whole blood was collected and CD3+, CD4+ and CD8+ cell counts and percentages were determined by flow cytometry at baseline and at 16 weeks, among 56 subjects who were included in the analyses. Results: A statistically significant increase in CD3+% in the 2000 IU/day vitamin D3 supplementation group, and increases in CD4+% in the 2000 IU/day and 4000 IU/day vitamin D3 supplementation groups were observed (p-values < 0.05) from the changes in baseline to 16 weeks. Further adjustments for age, sex and BMI showed that 2000 IU/day vitamin D3 supplementation increased in CD3+ count, CD3%, CD4 count, and CD4%, as compared to the placebo group (p-values < 0.05). Moreover, the highest serum 25(OH)D quantile group had the highest CD3% and CD4%. Conclusions: Sixteen-week vitamin D3 supplementation increases peripheral blood T-cell numbers and percentages in overweight/obese Black patients with vitamin D insufficiency. This resulting shift in circulating T-cell composition, particularly the increase in T helper cells (CD4+ cells), suggests that vitamin D supplementation may improve immune function in Black individuals.

Voluntary Wheel Running Reduces Hepatic Dendritic And Cytotoxic T Cells In Western Diet-fed C57Bl/6 J Mice

The activation of innate immunity and recruitment of infiltrating macrophages are critical components of non-alcoholic steatohepatitis (NASH). In mice, voluntary wheel running (VWR) has improved hepatic steatosis and markers of inflammation; however, few studies have evaluated the effects of VWR on hepatic macrophages and hepatic immune cells linked to the development of NASH. PURPOSE: To determine the preventative effects of voluntary wheel running on hepatic macrophages and hepatic immune cells in mice fed a Western diet for 18 weeks. METHODS: Male, 8-week old, C57Bl/6 J mice (n = 56; 14/group) were randomly assigned to either a normal diet (ND) or Western diet (WD; 45% lard, 17% fructose, 0.75% cholesterol), followed by again randomly dividing each dietary group into either a sedentary (SED) or VWR group. Following 18-weeks, the relative percentage of hepatic macrophages (F4/80intCD11b+Ly6C- Kupffer cell, F4/80intCD11b+Ly6C+ infiltrating macrophage, and F4/80+CD11b+CD11c+ dendritic cell) and hepatic immune cells (CD45+CD19+ B-cell, CD45+CD4+ T-cell, and CD45+CD8+ cytotoxic T-cell) were isolated and cell populations were identified by flow cytometry. Differences between the four groups (ND + SED, ND + VWR, WD + SED, and WD + VWR) were identified by one-way ANOVA. RESULTS: No significant differences in either macrophage or immune cell populations were observed between ND + SED and ND + VWR. When compared to ND + SED, WD + SED had significantly (p < 0.05) higher percentage of cytotoxic T-cells (3.4 ± 0.5% vs. 8.2 ± 1.9%), dendritic cells (2.1 ± 0.4% vs. 8.6 ± 2.0%), and infiltrating macrophages (0.6 ± 0.5% vs. 1.6 ± 1.9%). In contrast, WD + VWR had lower percentages of cytotoxic T-cells (3.9 ± 0.5%) and dendritic cells (5.4 ± 1.1%) such that the percentage of cells were not significantly different than ND + SED and ND + VWR. Infiltrating macrophages remained relatively unchanged (1.4 ± 0.2%) in WD + VWR. Despite the positive changes in WD + VWR, the percentage of cytotoxic T-cells (p = 0.056) and dendritic cells (p = 0.387) were not significantly different than WD + SED mice. CONCLUSIONS: In mice fed a Western diet for 18 weeks, voluntary wheel running reduced the relative percentage of dendritic cells and cytotoxic T cells in the liver. Supported by SIUE Seed Grants for Transitional and Exploratory Projects.

Residual Bone Marrow T & NK-Cells at Diagnosis in Pediatric Pre-B-ALL: A Case-Control Study.

BackgroundMature bone marrow T lymphocytes and NK may have a special relevance in the control of the malignant growth.ObjectiveWe aimed to assess the percentage of the residual BM T-cells, (T-helper –T-cytotoxic- NKT) and the NK cells of childhood precursor B-lymphoblastic leukemia (B-ALL) as an indicator of innate and adaptive immunity in these patients.Subjects and MethodsThis study was conducted on 40 B-ALL patients, and 40 apparently healthy matched children served as a control group. The flow cytometry was used to assess the percentage of the residual BM T-cells (T-helper, T-cytotoxic and NKT), and the NK cells.ResultsCompared with the control group, the percentage of the residual BM T-cells, its subtypes (T-helper, T-cytotoxic), and NKT cells in addition to the NK cells was significantly decreased in Group IA, and Group IB, but there was no significant difference between Group IA and Group IB in all studied parameters. In terms of the CD4/CD8 ratio, there was a significant increase in Group IA as compared to the control group (P < 0.026), but there were no significant statistical differences in CD4/CD8 ratio between Groups IB, and the control. Likewise, in CD4/CD8 ratio between groups IA, and Groups IB (P > 0.05). The percentage of NK, and NKT cells shows a significant increase in Hepatomegaly and Splenomegaly, as compared to non-Hepatomegaly and non-Splenomegaly patients of Groups IB (P < 0.05). However, there was a significant increase in statistical differences in the percentage of NKT cell between non-Splenomegaly, as compared to Splenomegaly patients of Group IA (P < 0.05). Additionally, there is a negative correlation between B.M Blast% to CD4/CD8 ratio and NK%, but there is no significant correlation between B.M Blast% to NK T% in the group 1 A.

Immunologic features of asymptomatic postvaccination infections with the Delta variant of SARS-CoV-2 in adults.

BackgroundAsymptomatic infections may play an important role in severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) Delta variant transmissions. However, the immunologic features of asymptomatic postvaccination infections with the Delta variant of SARS‐CoV‐2 in adults remain to be defined.MethodsA retrospective study involving 36 vaccinated adults infected with the SARS‐CoV‐2 Delta variant was performed. Their demographic and laboratory data were collected and analyzed in The First People's Hospital of Jingmen from August 4 to 20, 2021.ResultsOf the 36 adults, 6 persons had an asymptomatic infection. The severity of the SARS‐CoV‐2 infections was highly correlated with the doses of vaccinations (p = 0.019). The symptomatic and asymptomatic infected SARS‐CoV‐2 adults showed normal levels of leukocytes and lymphocytes. The C‐reactive protein (CRP) and interleukin‐6 (IL‐6) levels were elevated in the symptomatic groups. The period between the last vaccination to the time of infection in the asymptomatic group was longer than that in the mild and moderate groups (73 vs. 61 vs. 50 days; p = 0.047). The percentage of suppressor T‐cells in the asymptomatic group was the highest (32.2 ± 4.0% vs. 22.0 ± 7.2% vs. 29.3 ± 8.0%; p = 0.004). The signal‐to‐cutoff ratio value of total antibody against SARS‐CoV‐2 in the asymptomatic group was lower than that in the other two groups (383 vs. 703 vs. 1792; p < 0.001) and much lower than that in the moderate group. The multivariate ordinal logistic analysis after adjusting for gender, vaccination date, and vaccination dose indicated that CRP at Days 4−7 and 8−14, IL‐6 on Days 4−7, and total antibody were risk factors for coronavirus disease 2019 severity.Conclusions Asymptomatic postvaccination infections with the Delta variant of SARS‐CoV‐2 in adults tend to infect persons vaccinated twice. The immunophenotype profile for asymptomatic postvaccination infections is less inflammatory and accompanied by relatively lower antibody titers.

Abstract 575: Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors

Abstract In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p&amp;lt;0.001) when compared to TIL expanded in normoxia (20% O2). Collectively, these results support the advantage of adapting TIL to hypoxic conditions in the production of tumor-reactive TIL. Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 575.

Abstract 2097: Characterization of mouse and cellular phenotype of ABI1 KO T-cells

Abstract Background: The immune system plays a critical part in cancer development and progression. The increase in our understanding of mechanisms regulating immune-mediated cancer eradication has led to the development of immune-based therapies which have revolutionized treatment of some previously lethal malignancies. T-cell activation requires proper intercellular interactions mediated by cytoskeletal rearrangements and adhesion molecules, along with intracellular signal transduction via phosphorylation by tyrosine kinases (Lck and Fyn). Abelson interactor-1 (ABI1) is an adaptor protein known to regulate actin polymerization, adhesion molecules and tyrosine kinases. Previously, ABI1 has been shown to localize to the immunological synapse in an antigen-dependent manner and has been linked to regulation of IL-2 secretion. Published data from our lab shows that ABI1 has high affinity binding to SH2 domains of Src family kinases, Fyn and Lck, both of which are essential in T-cell activation. Additionally, ABI1 has been linked to hyperactivation of SFKs/STAT3/NF-kB signaling. In this study, we aim to delineate the role of ABI1 in T-cell activation, differentiation and signaling. Methods: We have generated a novel conditional knock-out mouse model to evaluate the effect of a T-cell specific ABI1 KO at the organismal level (ABI1(fl/fl)CD4Cre (ACD4). T-cells isolated from control (Abi1(fl/fl)CD4Cre(-)) and ABI1 KO mice (Abi1(fl/fl)CD4Cre(+)) were used for ex-vivo analysis and further characterization of ABI1 KO cellular phenotype with: Flow Cytometry (CD3+ subpopulations in immune organs), Immunofluorescence (actin polymerization), Western blotting (TCR-mediated T-cell activation) and RNAseq (transcriptional reprogramming). Results: In vitro studies of ABI1 KO primary murine CD3+ T-cells showed increased levels of NF-kB and STAT3. Flow cytometry analysis of CD3+ T cells subpopulations showed a difference in the percentage of CD4+ and CD8+ T cells; percentage of CD8+ T cells was increased, while percentage of CD4+ T cells was decreased in ABI1 KO vs. WT control mice. Furthermore, RNA seq analysis of CD8+ ABI1 KO T cells showed a significant decrease in the mRNA expression of Thpok (Zbtb7b) compared to WT. Conclusion: Our preliminary studies show that ABI1 is a potential modulator of T-cell activation through regulation of Lck/Fyn. This suggests ABI1 may play as a molecular switch negatively regulating T-cell activation by connecting phosphorylation-dependent signal transduction and actin cytoskeletal dynamics. Suggesting ABI1 as a new therapeutic to improve efficacy and longevity of T cell-based cancer immunotherapies. In addition, ABI1 appears to be implicated in the regulation of the percentage of CD4 and CD8 T-cells, a novel discovery which warrants further investigation. Citation Format: Maria Altagracia Ortiz, Andras Perl, Marcin Kortylewski, Leszek Kotula. Characterization of mouse and cellular phenotype of ABI1 KO T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2097.

Novel immunoprofiling method for diagnosing SLE and evaluating therapeutic response

ObjectiveDiagnosis of SLE is based on clinical manifestations but is heterogeneous in early onset. Hence, we aimed to evaluate the feature of the immunoprofiling in patients with SLE and apply...

Reference Intervals for Absolute and Percentage CD4+ T Lymphocytes among an Apparently Healthy Population in Addis Ababa, Ethiopia

BackgroundReference intervals for clinical laboratory parameters differ based on several factors, including age, sex, genetic variation, and geographic location. This variation influences clinical decisions and treatment monitoring. Currently, Ethiopia has used adopted reference intervals from manufacturer values derived from non-Africans. Therefore, the aim this study was to determine reference intervals for absolute and percentage CD4+ T cells for an apparently healthy population in Addis Ababa, Ethiopia.MethodsA community-based cross-sectional study was conducted on 361 apparently healthy people in four subcities in Addis Ababa from January to June 2019. Sociodemographic and clinical data were collected using a structured questionnaire after informed consent had been obtained. Blood samples were collected and CD4+ T-lymphocyte enumeration performed using a BD FACSPresto near-patient CD4 counter. Data were entered and analyzed using SPSS 20. Reference intervals were determined by a nonparametric test estimating percentiles 2.5 (lower limit) and 97.5 (upper limit) with 95% CIs. P<0.05 was considered statistically significant.ResultsA total of 337 (183 female and 154 male) healthy participants of median age 28 (IQR 17–35) years were included in the final analysis. Medians of absolute and percentage CD4+ T-cell counts (932.0 and 42.9, respectively) of female participants were significantly higher than male participants (802.5 and 38.7, respectively; P<0.05). Reference intervals for absolute CD4+ T-cell count and percentages in males were 483.8–1,310 cells/µL and 18.1–57.3 and in females 447.8–1,479.8 cells/µL and 25.6–58.9, respectively.ConclusionThe CD4+ T-count reference intervals established in this study showed some inconsistency from the manufacturer’s provided values and other studies and also revealed sex differences, necessitating sex-specific locally established reference intervals.

Intravenous calcitriol treatment benefits the homeostasis of CD4+ T cells and attenuates kidney injury in obese mice complicated with polymicrobial sepsis

ObjectivesSepsis is a lethal clinical condition with dysregulated cluster of differentiation (CD) 4+ T cells that leads to inflammation and multiorgan injury. Low vitamin D levels are commonly seen in patients with sepsis. Obesity is a state with oxidative stress and chronic inflammation. Both obesity and low vitamin D levels are associated with adverse outcomes in patients with sepsis. This study investigated the effects of calcitriol on CD4+ T-cell polarization and kidney injury during sepsis. MethodsMice were fed a high-fat diet to induce obesity. One group of obese mice served as the control group and in the other two groups (SS and SD) were performed cecal ligation and puncture (CLP) to induce sepsis. Mice in the SS group were injected with saline and those in the SD group with calcitriol 1 h after CLP via tail vein. Mice with sepsis were euthanized at 12 h and 24 h after CLP, respectively. ResultsSepsis led to a decrease in circulating CD4+ T-cell percentage, and T helper (Th) 2, Th17, and regulatory T (Treg) cell percentages were upregulated. Compared with the SS group, the SD group maintained blood CD4+ T-cell levels, and were reduced the Th2 and Th17 percentages as well as the Th17:Treg ratio. Also, plasma levels of cathelicidin increased, but inflammatory chemokines and kidney injury markers were reduced. Higher arginase-1 and lower inducible nitric oxide synthase expressions in the SD group indicated M1 macrophage polarized toward the M2 type. ConclusionsThese findings suggest that intravenous calcitriol administration after sepsis modulates the homeostasis of CD4+ T-cell subpopulations associated with alleviating sepsis-induced kidney injury in obese mice.

Impact of CD4+ T-cell count on sustained virologic response to direct-acting antivirals in hepatitis C virus monoinfected cancer patients: a prospective observational study

Limited data are available on the use of CD4+ T-cell count and percentage to predict response to direct-acting antiviral (DAA) treatment outside the hepatitis C virus (HCV)-HIV coinfected population. We sought to determine the impact of CD4+ T-cell count and percentage on response to DAAs in cancer patients with HCV monoinfection. Patients treated with DAAs were enrolled in a prospective observational study. CD4+ T-cell count and percentage was measured at baseline, end of treatment (EOT), and 12 weeks after the EOT (SVR12). A total of 174 patients were enrolled. Most patients (155/174, 89%) achieved an SVR12. A multivariate logistic regression model found that patients with hepatocellular carcinoma, HCV-3 and previous DAA treatment were more likely to develop treatment failure. Neither univariate analysis nor multivariate logistic regression analysis did show any association between CD4+ T-cell count or percentage and SVR12. CD4 T-cell count or percentage does not appear to impact SVR rates in cancer patients with HCV monoinfection receiving DAAs.