T-cell Surface Markers Research Articles

Abstract 4996: Analysis of the effect of eribulin on tumor immunity against triple-negative breast cancer

Abstract Purpose: Chemotherapeutic agents may induce immunomodulatory effects on immune cells and cancer cells in patients with triple negative breast cancer (TNBC). Recent studies have demonstrated that absolute lymphocyte count in peripheral blood can predict response to eribulin in patients with breast cancer, suggesting that eribulin have some favorable effects on immune cells. However, there have been no reports demonstrating the mechanisms of how eribulin affects the immune cells of patients with breast cancer. This study aimed to analyze the effect of eribulin on T cells in vitro and in vivo. Materials & Methods: Using human peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with TNBC, we investigated the effects of eribulin on proliferation of these cells, changes in the expression of surface markers, and antitumor effects on TNBC cells. Three TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-157) were used in this study. Human PBMCs were activated by CD3/CD28 stimulation in vitro for six days and analyzed for T-cell surface markers by flow cytometry. For three patients with TNBC who underwent eribulin treatment, blood was collected at baseline and on days 8, 22, and 64 after treatment, and PBMCs were isolated, and analyzed by flow cytometry. Results: The proliferation of activated CD8+ T cells from healthy donors and patients was facilitated when cultured with 1nM of eribulin compared to the control. Moreover, eribulin significantly decreased CD4/8 ratio in T cells (p< 0.05). In PBMCs from healthy donors, the frequency of CD45RA+ CD45RO- cells and CCR7+ cells in CD8+ T cells was significantly increased by eribulin treatment (p< 0.05). Furthermore, eribulin significantly increased CD127+ KLRG1- cells (memory precursor effector cells) and TCF1+ cells in CD8+ T cells (p< 0.01). Furthermore, the anti-tumor effect of activated T cells from PBMCs of healthy donors and patients with TNBC was enhanced when cultured with eribulin in all three TNBC cell lines compared to control, and IFNγ+ CD8+ T cells were significantly increased by eribulin treatment (p< 0.05). However, in TNBC patients treated with eribulin in clinical practice, there were no significant changes in the CD4/8 ratio, the frequency of CD45RA+ cells or CCR7+ cells in CD8+ T cells before and after treatment of eribulin. Conclusions: Our findings suggest that eribulin can facilitate the proliferation of CD8+ T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells in vitro. Further analysis of the effects of eribulin on TNBC cells and immune cells in vivo is required, and we continue to analyze immunomodulation in patients treated with eribulin. Citation Format: Shimizu Tadafumi, Takaaki Oba, Ken-ichi Ito. Analysis of the effect of eribulin on tumor immunity against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4996.

Varnimcabtagene Autoleucel (IMN-003A): Pharmacokinetic Profile with Predominant Naïve and Central Memory Phenotype Demonstrates Sustained In Vivo Persistence and Durable Responses in a First-in-India Industry Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies (RR BCM). A fractionated infusion of 1 x 10 6 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 10 6 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days as 10%, 30% and 60% fractions after Fludarabine-Cyclophosphamide lymphodepletion regimen (LR). Here we present var-cel pharmacokinetic data and correlation with efficacy. Methods: Varnimcabtagene autoleucel was manufactured using a cGMP compliant closed system (CliniMACS Prodigy ®). The manufacturing data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). Peripheral blood (PB) samples were obtained after consent at screening and as per patient schedule after infusion. Persistence of IMN-003A was evaluated in PB after infusion by ddPCR. Safety and efficacy data were collected for analysis. Bone marrow (BM) MRD (B-ALL) and PET-CT (B-NHL) was assessed at screening, D+28 and D+90 for efficacy. Minimal residual disease (MRD) was performed by FC with 10 -4 sensitivity. Apheresis, FP, and PB/BM samples on D+28 were analyzed for T-cell subpopulations and surface markers by FC. Statistical analysis was calculated using appropriate tests. Results: Of 25 patients (pts) enrolled and apheresed, 24 pts received var-cel (1 withdrawal) with majority of infusions on Days 0, +3, +7. Median product doubling time and manufacturing time was 1.04d (range 0.74-2.12) and 14 days (range 10 - 27) with 100% success with T cell purity median 99.4% (range 96.3-99.9). The mean % transduction of var-cel was 33.12% (range 8.50 - 58.35) with median transgene copies / genome of 2.57 (range 0.76 - 4.16). The mean CD4/CD8 ratio at apheresis and final product was 0.47 and 2.58 respectively. There was reversal of CD4/CD8 ratio from apheresis to final product (CAR+ T cells) which was statistically significant (p<0.01). There was statistically significant increase (p<0.01) in the number of naïve (CCR7+ RA+) and central memory (CCR7+ RA-) T cell subsets between apheresis and final product (CAR+ T cells). There was predominance of naïve and central memory CAR+ T cell subsets in the FP (58.19%; Figure 1A). Median Apheresis to Infusion time was 25d (range 16 - 95). There was a statistically significant trend (p<0.01) towards reversal of CD4/CD8 ratio in the PB from D0 to D+90. Var-cel showed maximum PB expansion (T max) at D+10 (range 7 - 28 days) with median C max 125,242 CAR copies / µg gDNA (range 18256 - 413,968). Among pts with 28-day and 90-day and last follow-up, 79.2%, 29.2% and 20.8% respectively had evidence of var-cel in PB, with median persistence of 28 days (range 10 - NR). Post-infusion, peak interleukin-6 level was median 42.3 pg/mL (range 6.71 - 17,481). B cell aplasia was observed in all pts concurrent with CAR-T expansion (Figure 1B). Median loss of B cell aplasia was not reached (range 42 - NR), with no apparent association with response at D+28 and D+90. Hypogammaglobulinemia was noted in 11 pts and 16 pts received IVIg. Of evaluable patients with CD4+ T-cell counts below 200/μL, recovery (>200/μL) was seen at median of 12 days (range 2 - NR) after var-cel infusion. Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%, MRD neg 83.3% (n=10/12); B-NHL 91.7%, CR 66.7%) and 80.9% (17/21) at D+90 [B-ALL 80%, MRD neg 80% (n=8/10); B-NHL 81.8% (9/11), CR 63.6%]. One B-NHL pt in PR at D+28 achieved CR at D+90. Three pts relapsed by D+90. Updated results will be presented at the meeting. Conclusion: Varnimcabtagene autoleucel manufacturing process results in a drug product characterized by highly pure T cells, with high proportion of naïve and central memory polyfunctional cells. In this industry-led first-in-India phase-2 study, var-cel resulted in a cytokine response with sustained in vivo proliferation and persistence resulting in robust and durable responses.

CLL Cell-Derived Exosomes Impair Immune Function and Normal Hematopoiesis in CLL

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms. Exosomes are nanovesicles that participate in bidirectional communication between neoplastic cells and their microenvironment. Using electron microscopy, we found that CLL cells produce and release CD19 +/CD5 +exosomes, and in agreement with other investigators, we detected high levels of CLL cell-derived exosomes in the plasma of patients with CLL. By using bead-assisted flow cytometry, we found that at least 50% of circulating extracellular vesicles are CLL cell-derived exosomes, identified as CD63 +/CD41a - particles co-expressing CD19 +/CD5 +, whereas only a small proportion of CLL patients' plasma-derived exosomes expressed T-cell or endothelial cell surface markers. Furthermore, we found a positive correlation between levels of CLL cell-derived exosomes in the plasma of patients with CLL and the patients' peripheral white blood cell counts, indicating that CLL cells are the primary source of circulating exosomes. To investigate the effects of CLL cell-derived exosomes, we conducted an analysis of CLL cells and CLL cell-derived exosomes from a cohort of 78 previously untreated patients. To identify the molecular profiles of CLL cell-derived exosomes, we analyzed their RNA transcripts using RNA-seq and qRT-PCR. Our data revealed that CLL cell-derived exosomes expressed significant levels of oncogenic micro-RNAs. Additionally, we identified 1052 differentially expressed genes, and enrichment analysis further unveiled the significant upregulation of gene clusters associated with p53, NF-κB, MAPK, mTOR, and interleukin signal transduction pathways, consistent with their neoplastic cell origin. The distinctive transcriptional profile of CLL cell-derived exosomes led us to explore their effects on normal cell populations. We labeled ultra-centrifuged CLL cell-derived exosomes with fluorescent markers and incubated them with various cell types in culture conditions conducive to their physiological activity. Using confocal microscopy and flow cytometry, we observed significant uptake of CLL cell-derived exosomes by normal monocytes, fibrocytes, B cells, T cells, and CLL cells. Furthermore, incubation with CLL exosomes resulted in notable alterations in normal CD14 + monocyte polarity and subset-specific shifts in the expression of inflammatory cytokines. Remarkably, CLL cell-derived exosomes induced overexpression of the T-cell makers CD5 and CD8 in normal CD19 + cells, suggesting that CLL exosomes may promote immune-evasive reprogramming of normal B cells. Moreover, when normal T cells were incubated with CLL cell-derived exosomes, CyTOF analysis using 46 markers revealed elevated levels of immune checkpoints, including PD1, CTLA4, and LAG3, in both CD4 + and CD8 + subsets, indicating that exosomes derived from CLL cells induce T-cell dysfunction. Given their profound effect on immune cells, we wondered whether CLL exosomes affect hematopoietic cells. We previously found that normal monocyte derived fibrocytes stimulate hematopoietic colony proliferation. In sharp contrast to control fibrocytes, fibrocytes preincubated with CLL exosomes and CLL cell-derived exosomes inhibited normal BFU-E and CFU-GM colony formation, suggesting that CLL exosomes might contribute to CLL patients' anemia or neutropenia. Wondering whether CLL cell-derived exosomes affect the viability of other cell types, we found that they induced a dose-dependent apoptosis of CD19 + B cells, CLL cells but not normal T cells. In summary, CLL cell-derived exosomes exert a profound effect on gene and cell surface protein expression in immune and tissue-remodeling cells, leading to functional modifications, alteration of inflammatory cytokine levels, and suppression of normal hematopoiesis. The impact of CLL exosomes may vary across different cell types; however, as the leukemic cell mass increases, exosome levels escalate, invariably affecting CLL cell environment. The identification of exosomes with distinct molecular signatures holds promising potential for novel therapeutic interventions in future treatment of CLL.

Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

Response, Peak and Persistence of Varnimcabtagene Autoleucel (IMN-003A), First-in-India Industry CD19-Directed CAR-T Cell Therapy, with Fractionated Infusions for Patients with Relapsed and/or Refractory B Cell Malignancies: Early Results (IMAGINE Study)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, which is being tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies (RR BCM). A fractionated infusion of 1 x 106 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 106 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days (D0, D+1, D+2) as 10%, 30% and 60% fractions after Fludarabine-Cyclophosphamide lymphodepletion regimen (LR). Safety and efficacy results are submitted in a separate abstract (#166297); here we present the early IMN-003A pharmacokinetic data and correlation with disease response. Methods: IMN-003A was manufactured using cGMP compliant closed system (CliniMACS Prodigy®). The manufacturing data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). Peripheral blood (PB) samples were obtained after consent at screening and as per patient schedule after infusion. Persistence of IMN-003A was evaluated in PB after infusion by ddPCR. Safety and efficacy data were collected for analysis. Bone marrow (BM) samples for MRD were obtained at screening, D+28 and D+90 for efficacy. Minimal residual disease (MRD) was performed by FC with 10-4 sensitivity. Apheresis, FP, and PB samples were analyzed for T-cell subpopulations and surface markers by FC. Results: Eight apheresis were obtained and transduced to achieve the final product with a median manufacturing time of 11d (range 10-18) with 100% success. The mean % transduction of IMN-003A on autologous T cells was 34.77% (range 15.49 - 58.35) with median transgene copies / genome of 2.25 (range 1.49 - 3.83). The mean CD4/CD8 ratio at apheresis and final product was 0.74 and 0.84 respectively. The T-cell subpopulations at apheresis and FP were analyzed (Figure 1A). The mean proportion of CD4 and CD8 positive naïve cells (CCR7+ RA+) was above 90% in the final product (Figure 1A). Median Product doubling time and Apheresis to Infusion time was 1.04d (range 1.02 - 1.38) and 20d (range 16 - 30) respectively. Post IMN-003A infusion, there was reversal of CD4/CD8 ratio in peripheral blood (Figure 1A). IMN-003A CAR-T cells showed maximum PB expansion (Tmax) at median 10d (range 10 - 14) with median Cmax 180,680 CAR copies / µg gDNA (range 94,675 - 276,412). Among pts with 28-day follow-up, 80% had evidence of CAR+ T cells in PB, with median persistence not reached (range 28 - NR). B-cell aplasia was observed in all pts concurring with CART expansion (Figure 1B). Median loss of B cell aplasia was not reached (range 42 - NR), with no apparent association with response at D+28 and D+90. Hypogammaglobulinemia was noted in 6 of 7 evaluable pts and 2 pts received IVIg. Of evaluable patients with CD4+ T-cell counts below 200/μL, recovery (>200/μL) was seen at median of 10 days (range 2 - 10) after the IMN-003A cell infusion. None of these pts have relapsed. Of 5 MRD-evaluable pts, response at day +28 (n=4) and +90 (n=1) was 100%. Updated results will be presented at the meeting. Conclusion: In this industry-led first-in-India phase-2 study, IMN-003A production in a cGMP closed system was feasible and rapid. The peak of CAR-T expansion occurs at median 10d of infusion. The interim responses obtained are deep. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 6120: The effect of chronic stress in T-cell population and function in ovarian cancer

Abstract Ovarian cancer (OC) is the deadliest form of gynecological malignancies highlighted by a five-year survival rate of less than 49%. Late diagnosis and chemoresistance represent the main hurdle in the effort to improve OC patient outcomes. Currently, immunotherapy is studied as an alternative to front-line therapy, but its use in the clinic remains limited due to its modest impact on patient outcomes. It is well known that the neuroendocrine system can modulate immune cell behavior. This study aims to determine how behavioral factors, such as chronic stress and an associated increase in stress hormone levels, impact T-cell infiltration and function in ovarian cancer. As T-cells play a central role in cancer recognition and killing, we hypothesize that stress hormones could decrease the efficacy of checkpoint blockade by inducing changes in T-cell populations and contribute to T-cell exhaustion (determined by the expression of PD-1, TIM-3, FOXP3, and CTLA-4). We obtained tumor samples from C57/BL6 ID8 tumor-bearing mice subjected to daily restraint stress to dissect the interaction between the neuroendocrine system and tumor-associated T-cells. Unstressed mice were used as controls. Tumors from both groups were collected and preserved with paraffin for histological assessments. Immunohistochemistry analyses were used to evaluate the presence of CD4+ and CD8+ T-cells. To measure specific CD4+ T-cell subtype, regulatory T-cells (T-reg), FOXP3 was used as a marker. In addition, we assessed for T-cell surface markers PD-1, CTLA-4, and TIM-3. Results show a significant increase in PD1+ expressing cells (2.35x-fold; p= 0.0173) in tumors from the stress group. On the other hand, there was a non-significant decrease in CD4+ T-cells (0.78x-fold; p=0.4286) and non-significant increase in CD8+ T-cells (1.75x-fold; p= 0.1833) between groups. Although we did not observe significant differences in FOX-P3 (1.4x-fold; p=0.3152), we did observe a trend towards increased expression of this transcription factor, suggesting a predominance of T-regs in the tumor microenvironment. When comparing the presence of surface markers, there was a non-significant increase in TIM-3 (1.56x-fold; p=0.5273) and CTLA-4 (1.32x-fold; p=0.5303) positive cells in the tumor microenvironment of samples from the stress group. These data suggest that the neuroendocrine system may modulate T-cell activity, specifically the expression of PD-1 ligand. Furthermore, sustained release of stress hormones may promote an immunosuppressive tumor microenvironment, leading to T-cell exhaustion, immune evasion, and tumor progression. Citation Format: Camily Morales-López, Alexandra Aquino-Acevedo, Melanie E. Cruz-Robles, Melanie Ortiz-León, Elvin R. Hernández-Cordero, Yadiel A. Rivera-López, Margarita Rivera-Bonilla, Rebecca A. Previs, Guillermo N. Armaiz-Pena. The effect of chronic stress in T-cell population and function in ovarian cancer [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 6120.

Novel T Follicular Helper-like T-Cell Lymphoma Therapies: From Preclinical Evaluation to Clinical Reality.

Simple SummaryThis work reviews the multiple efforts that have been and are being invested by researchers as well as clinicians to improve the treatment of a specific T-cell lymphoma called follicular helper peripheral T-cell lymphoma. Still, though treatments for B-cell lymphomas have improved, this particular T-cell lymphoma has little to no new therapeutic options that show marked improvements in the survival of the patients compared to treatment with chemotherapy. We report here the evaluation of targeted new therapies for this T-cell lymphoma in new preclinical models for this cancer or in clinical trials with the objective to offer better (combination) treatment options.The classification of peripheral T-cell lymphomas (PTCL) is constantly changing and contains multiple subtypes. Here, we focus on Tfh-like PTCL, to which angioimmunoblastic T-cell lymphoma (AITL) belongs, according to the last WHO classification. The first-line treatment of these malignancies still relies on chemotherapy but gives very unsatisfying results for these patients. Enormous progress in the last decade in terms of understanding the implicated genetic mutations leading to signaling and epigenetic pathway deregulation in Tfh PTCL allowed the research community to propose new therapeutic approaches. These findings point towards new biomarkers and new therapies, including hypomethylating agents, such as azacytidine, and inhibitors of the TCR-hyperactivating molecules in Tfh PTCL. Additionally, metabolic interference, inhibitors of the NF-κB and PI3K-mTOR pathways and possibly novel immunotherapies, such as antibodies and chimeric antigen receptors (CAR) directed against Tfh malignant T-cell surface markers, are discussed in this review among other new treatment options.

Correlative Biological Studies Related to the Response, Peak and Persistence of ARI0002h, an Academic BCMA-Directed CAR-T Cell, with Fractionated Initial Infusion and Booster Dose for Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Correlative Biological Studies Related to the Response, Peak and Persistence of ARI0002h, an Academic BCMA-Directed CAR-T Cell, with Fractionated Initial Infusion and Booster Dose for Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Divergent Immune Responses to SARS-CoV-2 Vaccines in Immunocompromised Patients.

To The Editor: Understanding the composition and duration of immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is critical for prevention of infection. The most critical elements of immunity to SARS-CoV-2 are neutralizing antibody and T-cell immunity. Current assessments of immunity and risk for infections largely depended on detection of antibodies to SARS-CoV-2. However, taken alone, this represents an often-unreliable tool due to the evanescent nature of spike immunoglobulin G (IgG) responses.1-3 Here, an important and more durable response involves cytotoxic T-cells that can eliminate virally infected cells and T helper cells, which are critical to coordinating adaptive immunity toward the virus and generating long-lasting immunologic memory. However, T-cell responses are more difficult to assess. These issues become more prescient in determining immunity to SARS-CoV-2 in immunocompromised individuals where a majority show no IgG responses to vaccines and the recent emergence of the Delta variant with reports of >74% vaccine breakthrough cases.4,5 Here, we report on patients demonstrating divergent immune responses to SARS-CoV-2 vaccination. Institutional Review Board approval and informed consent was obtained before performance of assays (Appendix 1, SDC, https://links.lww.com/TP/C290). Seven patients were identified from a cohort of 70 immunocompromised patients, with all demonstrating spike-specific IgG unresponsiveness 2–4 mo postvaccination. All had received treatment with B-cell modifying agents. We subsequently examined CD4+/CD8+ T-cell–spike-specific immune responses in all patients and repeat examination after revaccination in 2 patients (see Appendix 1, SDC, https://links.lww.com/TP/C290, for Methods and Data). CD4+/CD8+ T-cell responses are shown in Figure 1 and demonstrate robust CD4+T-cell (0.94 ± 1.2%, Normal > 0.05%) and CD8+ T-cell (0.89 ± 1.2%, Normal > 0.05%) immune responses to spike peptides. Two patients receiving Johnson & Johnson booster vaccines demonstrated increased T-cell responses but remained spike IgG negative. This suggests that T-cell immune responses to SARS-CoV-2 vaccines are primal and retentive and that B-cell depletion before vaccine exposure prevents the cascade of progression of B-cell activation necessary for SARS-CoV-2 spike IgG production.FIGURE 1.: A, Detectable CD4+/CD8+ T-cell immune responses in vaccinated patients against SARS-CoV-2 spike peptides. Flow cytometry diagrams from 7 patients (P1–P7) assessed after 2 doses of Pfizer or Moderna vaccines. Cells were stimulated by SARS-CoV-2 spike peptides pool (PepMix SARS-CoV-2 [spike glycoprotein, JPT) for 9 h in vitro in the presence of Brefeldin A and anti-CD28/CD49d (BD Bioscience, San Jose, CA). Cells were stained for T-cell surface markers followed by fixation, permeabilization, and intracellular staining of cytokines (BD Bioscience, San Jose, CA). Activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) are shown in the upper right-hand corner of each flow box. B, This figure shows immune responses to SARS-CoV-2 spike peptides in 2 patients who obtained external revaccination with the Johnson & Johnson vaccine. As in (A), percentages of activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) in blood are shown after first vaccination (2 doses) and after third vaccination (booster). Activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) are shown in the upper right-hand corner of each flow box. IFNγ, interferon γ; IL, interleukin; P, patient; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNFα, tumor necrosis factor α.Absence of IgG responses to SARS-CoV-2 suggests patients be considered “unvaccinated.” However, we show that patients treated with B-cell–modifying agents develop robust T-cell immune responses to SARS-CoV-2 without generating IgG responses. These observations should be considered in light of data presented by Peng et al showing robust CD4+/CD8+ T-cell responses to SARS-CoV-2 after infection. A critical observation was the diversity of T-cell responses that likely extend beyond the persistence of spike antibody. IgG is necessary for sterilizing immunity and T-cells cannot prevent infection as antigen presentation is required, but T-cell immunity can be at the ready for viral elimination. This may provide an inside track for rapid deployment of SARS-CoV-2 immunity, and although not preventing infection, could alter the severity and duration of SARS-CoV-2 disease.6 This is supported by data from Oberhardt et al7 who recently showed that vaccine-induced CD8+ T cells were the main mediators of protection after vaccination since they emerged before detection of neutralizing antibody and expanded after booster vaccination. Thus, detection of T-cell immune responses in patients failing to generate spike-specific IgG may aid in a more comprehensive assessment of immunity to SARS-CoV-2, identifying patients who would no longer be considered “unvaccinated” based on negative spike-specific IgG. This likely has relevance to patients receiving B-cell directed therapies for autoimmune and hematologic diseases. ACKNOWLEDGMENTS The authors would like to express our gratitude to the members of the Transplant Immunotherapy Program and the Transplant Immunology Laboratory at Cedars-Sinai Medical Center for their dedication to improving patient outcomes and safety during the coronavirus disease 2019 pandemic. We would also like to thank the patients who participated in this study by donating their blood for analysis of SARS-CoV-2 reactive T-cells.

GD2-directed bispecific trifunctional antibody outperforms dinutuximab beta in a murine model for aggressive metastasized neuroblastoma

BackgroundNeuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal...

A T-Cell Surface Marker Panel Predicts Murine Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8+ T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.

Circulating MicroRNAs and T-Cell Cytokine Expression Are Associated With the Characteristics of Asthma Exacerbation.

PurposeImmunological mechanisms underlying asthma exacerbation have not been elucidated. The aim of this study was to assess the associations of various asthma exacerbation traits with selected serum microRNA (miRNA) expression and T-cell subpopulations.MethodsTwenty-one asthmatics were studied during asthma exacerbation (exacerbation visit [EV] and the follow-up visit [FV] at 6 weeks). At both visits, spirometry was performed, fractional exhaled nitric oxide (FeNO) was measured, and nasopharyngeal and blood samples were collected. In nasopharyngeal samples, respiratory viruses were assayed by multiplex polymerase chain reaction (PCR), and bacterial cultures were performed. Serum miRNAs were assayed with real-time PCR. T-cell surface markers, eosinophil progenitors and intracellular cytokines were assessed by flow cytometry.ResultsTwo-thirds of patients had moderate or severe exacerbation and the FV, overall improvement in asthma control was observed. The mean expression of serum miRNA-126a, miRNA-16 and miRNA-21 was significantly lower at the EV than at the FV. At EV, miRNA-29b correlated with FeNO (r = 0.44, P < 0.05), and 5 of 7 miRNA tested correlated with pulmonary function tests. The number of cluster of differentiation (CD)45+CD4+interleukin (IL)4+ cells was significantly higher at the EV than at the FV, and positive correlations of T-regulatory cells and eosinophil progenitors with asthma control was found. At the EV, serum miRNAs negatively correlated with the number of T cells expressing IL-4, IL-17, IL-22 and interferon gamma, while at the FV both positive and negative correlations with T-cell subsets were observed. No association of detected pathogen (viruses and bacteria) in nasopharyngeal fluid with clinical, functional and immunological parameters was found.ConclusionsEpigenetic dysregulation during asthma exacerbation could be related to respiratory function, airway inflammation and T-cell cytokine expression.

Association of PD-L1, PD-L2, and Immune Response Markers in Matched Renal Clear Cell Carcinoma Primary and Metastatic Tissue Specimens.

Immune checkpoint therapy has been promising in renal cell carcinoma, but no validated clinically relevant biomarkers exist. Metastatic deposits may have discordant biomarker expression. Fifty matched pairs of primary and metastatic kidney tumors were evaluated via immunohistochemistry for immune checkpoint proteins PD-1, PD-L1, and PD-L2 and the T-cell and macrophage surface markers CD3, FOXP3, and CD163. Semiquantitative scores incorporating prevalence of both tumor and nontumor labeling were compared between metastatic and primary kidney tumor specimens. A large minority of patients had discordant expression of PD-1 (31.2%), PD-L1 (22.5%), or PD-L2 (21.5%) between primary and metastatic sites. The expression of the novel marker PD-L2 correlated with both PD-1 (r = 0.47, P = .02) and PD-L1 (r = 0.67, P < .001) in metastatic deposits. This study demonstrates that renal clear cell carcinoma primary tumors and metastatic deposits have some discordance in the expression of PD-L1, PD-1, and PD-L2.

The effect of fluid resuscitation strategy on monocyte and T-cell surface markers

BackgroundDespite initial lifesaving benefits, posttraumatic resuscitation strategies have been associated with immunologic complications leading to systemic inflammatory response syndrome, sepsis, multiple organ failure, and late trauma death. Nevertheless, the direct effect on immunologic surface markers remains inadequately described. We hypothesized that changes in monocyte and T-cell surface markers were associated with initial posttraumatic fluid resuscitation. Materials and methodsData were extracted from the inflammation and host response to injury (Glue Grant) study. Blood samples were drawn from 492 patients on days 0, 1, 4, 7, 14, and 28 and analyzed for 31 monocyte and T-cell surface markers. Resuscitation strategies during the initial 48 h were quantified, including transfusion of packed red blood cells (PRBCs), fresh frozen plasma (FFP), platelets, and crystalloids. Longitudinal surface marker concentration changes were quantified by the calculation of a within-patient signal intensity change and were associated with resuscitation strategy while controlling confounders. P-values were post hoc corrected using the false detection rate q-value. ResultsThe monocyte surface marker (CD83) trajectory (as measured by a within-patient signal intensity change) was found to be positively associated with volume of PRBCs transfused (q = 0.002) and negatively associated with the transfused volume of FFP (q = 0.004). T-cell surface marker (CD3) was found to be negatively associated with volume of PRBCs transfused (q = 854 × 10−9) and positively associated with the transfused volume of FFP (q = 0.022). Platelets and crystalloid transfusion volumes were not associated with any surface marker trajectories. ConclusionsPRBC and FFP transfusion was associated with opposing effects on CD3 and CD83 trajectories, which may in part explain some of the protective effects of a high FFP:PRBC ratio in trauma-related resuscitation.

Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies.

T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell-T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB-mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. Cancer Immunol Res; 6(1); 47-58. ©2017 AACR.

HMGB1/anti-HMGB1 antibodies define a molecular signature of early stages of HIV-Associated Neurocognitive Disorders (HAND)

BackgroundHIV-associated neurocognitive disorders (HAND) persist in the post-HAART era, characterized by asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorders (MND). High mobility group box 1 (HMGB1) is a non-histone chromosomal protein widely expressed in the nucleus of all eukaryotic cells, including brain cells, which acts as a potent proinflammatory cytokine when actively secreted from immune cells. Recent reports suggested that HMGB1 acts on microglial cells to promote neuroinflammation. In this study, our aim was to determine whether HMGB1 is involved in HAND, but also to identify early new markers of neurological impairment in HIV-infected patients.MethodsCSF and serum were collected from 103 HIV-1-infected patients enrolled in Neuradapt, a prospective study of the prevalence of HAND in HIV-1 infected patients at Nice University Hospital. Stored fluids were assessed for immunological, virological, and brain metabolite parameters. In addition to HIV RNA and DNA measurements, expression of T-cell surface markers of activation (CD38 and HLA-DR) was analyzed on whole blood. Concentration of 27 cytokines and chemokines was measured using multiplex bead assays on serum and CSF. Concentration of HMGB1 and anti-HMGB1 IgG autoantibodies were also measured on the same samples. Changes in cerebral metabolites N-acetyl aspartate (NAA), Choline (Cho) and creatinine (Cr) were assessed by magnetic resonance microscopy (MRS).ResultsClinical, virological and immunological characteristics were comparable between HAND (n = 30) and no HAND (n = 73) patients, except the absolute numbers of CD8+ T cells, which were higher in patients with HAND. Among the 29 molecules tested, only 4 of them were significantly upregulated in the CSF from HAND patients as compared to healthy donors i.e. HMGB1, anti-HMGB1 IgG antibodies, IP-10 and MCP1. CSF HMGB1 levels were positively correlated with HIV-1 DNA in aviremic HAND patients, suggesting a positive impact of HMGB1 on HIV reservoirs. Moreover, in contrast to NAA/Cr and Cho/NAA ratios, circulating anti-HMGB1 IgG antibody levels could discriminate patients with no HAND from patients with no HAND and a single deficit (average ROC-AUC = 0.744, p = 0.03 for viremic patients), thus enabling the identification of a very early stage of neurocognitive impairment,ConclusionWe report that brain injury in chronically HIV-infected patients on stable HAART is strongly associated with persistent CNS inflammation, which is correlated with increased levels of HMGB1 and anti-HMGB1 IgG in the CSF. Moreover, we identified circulating anti-HMGB1 IgG as a very early biomarker of neurological impairment in patients without HAND. These results might have important implication for the identification of patients who are at high risk of developing neurological disorders.

Immune Response Profiling of Patients with Anogenital Warts

ABSTRACTThe incidence of anogenital warts, commonly caused by human papillomavirus (HPV-6 and HPV-11), is increasing worldwide. These infections are frequently associated with relapse, possibly due to weak host immunity. However, the role of cell-mediated immune players in combating infection is not clearly understood till date. Here, we attempted to understand the immune profile among patients with anogenital warts. In this study, we compared the T-helper cell (Th1 and Th2) response in patients with venereal warts due to HPV-6 and HPV-11 infection relative to healthy controls (HCs)in vitro. In thein vitromodel, the peripheral blood mononuclear cells were stimulated with HPV peptide 6 or 11, stained for T-cell surface marker and intracellular cytokines (interferon [IFN]-□ and interleukin [IL]-4), and were analyzed by flow cytometry. In the present study, significant decrease was observed in the frequency of IFN-□ T cells as compared with HCs. On the contrary, frequency of T cells expressing IL-4 was significantly increased in the patients. The observed functional skewing of HPV-specific T cells from Th1 to Th2 response in patients indicated suppressed immunity against the HPVs. Findings of our study have potential in the near future for strategizing adjunct immunomodulation approaches with the standard treatment for early remission and prevention of recurrence.How to cite this articleSingh M, Thakral D, Kar HK, Rishi N, Mitra DK. Immune Response Profiling of Patients with Anogenital Warts. Indian J Med Biochem 2017;21(1):11-16.

Multiparameter mass cytometry of T cell development and selection

Abstract T cell development and selection in the thymus play key roles in shaping the adaptive immune system and maintaining self-tolerance. Previous studies characterizing thymic development have largely relied on genetic perturbations and subsequent cell sorting that invariably perturb specific developmental compartments. Using mass-cytometry, allow us to capture the entire developmental progression of T cells, from the double-negative (DN) stages, through double-positive (DP) and the CD4 and CD8 branches, without perturbation. With 42 channels simultaneously measured, we collected a mass cytometry dataset profiling the mouse thymus with T-cell surface markers and transcription factors, chosen based on their broad functionality in T cell development. Using the newly developed “Wishbone” algorithm, we recovered the known stages in T cell development with high accuracy and developmental resolution. This allowed us to place DN, DP, CD4+ and CD8+ cells from a single thymus along a unified bifurcating trajectory. Our data allows for precise ordering of multiple events along the trajectory using un-sorted cells from mice that were not perturbed genetically. To characterize the selection process along the development trajectory we used Nur77-GFP mice in which GFP is induced by TCR signaling that occurs during positive and negative selection. By following GFP strength together with cell trajectory we were able to recognize and characterize the behavior of proteins that participate in selection and that are involved in the transition from the DP to SP stage. This approach enables us for the first time to place and follow coordinated changes in multiple markers that take part in T cell development and selection in the thymus using single snapshots

αβ T-cell receptor bias in disease and therapy (Review).

The diversity and specificity of T cell receptors (TCR), the characteristics of T-cell surface marker, are central to the adaptive immunity. TCR variability is required for successful immunization coverage because this structural foundation is indispensable for the valid identification of short antigen peptides (derived from degraded antigens) that are presented by major histocompatibility molecules on the surfaces of antigen-presenting cells. Despite the vast T-cell repertoire, biased αβ TCR has become a common theme in immunology. To date, numerous examples of TCR bias have been observed in various diseases. Immunotherapy strategies that are based on αβ T cell responses are also emerged as a prominent component of clinical treatment. In the present review, we briefly summarize the current knowledge regarding basic structural information and the molecular mechanisms underlying TCR diversity. Moreover, we outline the role of TCR repertoire bias in some diseases, and its application for therapeutic interventions, as these play significant roles in disease progression, even with patients with a good prognosis.