T-cell Populations Research Articles

In-Depth Immune Profiling in Children with BCP-ALL within the AIEOP-BFM ALL 2017 Study

The international multicenter frontline therapy trial AIEOP-BFM ALL 2017 prospectively randomizes CD19-targeting T-cell engager blinatumomab (BLN) against standard of care (SOC) to improve outcomes in pediatric patients (pts) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) stratified for high (HR) and medium (MR) risks of relapse. In HR arm BLN is given earlier, directly after intensified HR therapy block, while in the MR arm, it is given towards the end of chemotherapy, before maintenance. The immune composition prior to BLN might thus differ between both treatment groups (HR vs MR) which may impact BLN-mediated anti-leukemic activity. Within the Germany-wide immune monitoring program accompanying the AIEOP-BFM ALL 2017 trial we addressed this question and studied a total of 1056 longitudinal peripheral blood samples collected between 11/2018 and 05/2022 from 381 BCP-ALL pts. Samples were prospectively analyzed with multicolor flow cytometry to study immune cell composition (Panel 1) at the time of BLN application (d0) and under ongoing immunotherapy within first 24h (d1), at day 14 (d14), at the end of the cycle I (d28) and end of cycle 2 (day28_II), if applicable (Figure 1A). In-depth T-cell profiling was performed in the BLN arm, utilizing 4 additional 12-color panels concentrated on markers associated with T-cell functionality (measured with standardized cytometer settings). Data were analyzed using classical 2D-manual gating strategies (> 2000 variables) and completed with R-based machine learning algorithms; namely partial Least-Squares Discriminant Analysis (PLS-DA), a multivariate dimensionality-reduction tool for identifying HR- and MR-defining immune signatures and PhenoGraph algorithm for unsupervised T-cell cluster detection. T-cell receptor excision circles (TRECs) and TR-repertoire analysis were also performed among paired samples of 117 BLN-treated pts. Baseline immune composition differed notably between HR and MR pts directly prior to BLN therapy, as shown by PLS-DA analysis on manually gated immune subsets. HR pts showed higher neutrophil counts and lower lymphocyte and monocyte counts compared to MR pts. Interestingly, MR pts had higher levels of B- and T-cells, which are crucial for BLN-mediated T-cell activation. HR pts displayed an elevated CD4+/CD8+ ratio, shifted naïve/memory T-cell phenotypes and increased proportions of immunosuppressive T-regulatory cells. These results suggest that more intensive therapy protocols directly prior to BLN influence the immune composition towards compromised states in the HR arm, potentially affecting BLN-mediated T-cell leukemia control. BLN effectively depleted CD19+ cells in 221 out of 224 pts. We observed significantly stronger T-cell activation/expansion in MR pts compared to HR pts under BLN therapy. This expansion that was primarily attributed to naïve CD8+ T-cell populations, resulting from clonal T-cell proliferation independent of thymic contribution, as supported by TREC and TR-repertoire analysis. The PhenoGraph clustering also identified this expanding naïve CD8+ T-cell populations, surprisingly marked by loss of adhesion molecule DNAM1 (panel 1), normally involved in NK and T-cell mediated cytotoxicity and by gaining the expression of TIM-3 (panel 3), an inhibitory molecule usually co-expressed with PD1 on exhausted T-cells with memory phenotypes. In this setting however, TIM3 was not co-expressed with PD1 (Figure 1B) and its expression was correlated with the B-cell levels at baseline and was strongest in MR pts under BLN therapy. Our data suggest an until now undescribed association between DNAM1 and TIM3 with BLN-mediated T-cell activation, hinting that they may serve as a measure of T-cell activation strength or an early T-cell exhaustion marker. In conclusion our study provides clinically relevant insights in how chemotherapy prior BLN might affect the patients´ immune system, possibly influencing the BLN anti-leukemic activity, and helps to determine the optimal place of immunotherapy with BLN in multimodal treatment schedules. Here, we report until know undescribed effect of BLN on T-cell compartment. Upcoming clinical data, which will be correlated with obtained immune profiles will answer the open question regarding the clinical relevance of these findings, and shed light into the mechanisms determining response to BLN, for its maximum benefit in childhood ALL.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Dose Escalation of ISB 1342, a Novel CD38xCD3 Bispecific Antibody, in Patients with Relapsed / Refractory Multiple Myeloma (RRMM)

Introduction: ISB 1342 is a humanized CD38xCD3 bispecific T-cell engager using Ichnos proprietary Bispecific Engagement by Antibodies based on the T cell receptor (BEAT ®) platform, designed to simultaneously bind CD38 on multiple myeloma (MM) cells and CD3ε on T cells, to induce tumor killing. This mechanism of action is different from existing monospecific CD38 targeting therapies and was designed to overcome resistance to daratumumab in MM. ISB 1342 exhibits potent killing of the primary MM cells and MM cell lines with low sensitivity to daratumumab ( Pouleau B. et al. Blood 2023; 142 (3): 260-273). We report here, the updated findings from the weekly dose-escalations of intravenous (IV) and subcutaneous (SC) injections of an ongoing, single-agent phase 1 study (NCT03309111) of ISB 1342 in patients with RRMM. Methods: Adult patients with RRMM that relapsed after or were refractory to prior therapies, including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody; received ISB 1342 on a weekly (QW) schedule by IV or SC injection with a priming dose on the first day in 2 parallel dose-escalation schemas. Patients had measurable disease per the International Myeloma Working Group (IMWG) criteria (2016). Dose escalation in the QW schedule began at 0.2/0.3 mg/kg priming/treatment dose and followed a “3 + 3” design. The primary study objectives are to determine the maximum tolerated dose (MTD) and select a recommended phase 2 dose (RP2D) of ISB 1342. Secondary objectives includ evaluation of anti-myeloma activity, pharmacokinetics (PK), pharmacodynamics, and immunogenicity of ISB 1342. Results: As of July 18, 2023, based on ongoing clinical database, 39 subjects had received weekly IV infusions of ISB 1342 in 8 dose-escalation groups from 0.2/0.3 mg/kg to 4.0/16.0 mg/kg as priming/treatment dose; and 7 additional subjects had received weekly SC injections at 2.0/8.0 mg/kg. Two different formulations have been tested in SC group. Median number of cycles administered was 2. The majority were male (57%) and white (74%); 13% were Black or African American. The median age was 69 years (range, 54-76) and the median time since diagnosis was 6.6 years (range, 2.8-22.2). The median number of prior anti-myeloma lines of therapy was 6 (range 1-12). Twenty-one (67%) subjects were considered to be triple-class refractory and 18 (48%) penta-drug refractory. Forty-one (89%) subjects experienced treatment-related adverse events (TRAEs) of any grade. Most TRAEs were grade 1-2, including infusion related reactions (37%), cytokine release syndrome (CRS, 34%, all grade 1or 2), anemia (24%), neutropenia (24%), and thrombocytopenia (17%). Five subjects in SC group (71% of SC) had injection site reaction, all grade 1 or 2. Grade 3 or higher TRAEs occurring in more than 5% of subjects were infusion related reaction (no grade 4), anemia (no grade 4), neutropenia, leukopenia. No Grade 5 TRAE was observed. After QW IV infusion, ISB 1342 serum concentration peaked at the end of infusion and then showed bi-exponential decline. ISB 1342 serum exposures increased in a dose linear fashion across the tested IV infusion dose range. The limited PK data after SC injection suggest slow presentation of ISB 1342 into the systemic circulation leading to lower C max and delayed T max relative to IV. Transient increases in serum cytokines were observed within 24 hours after ISB 1342 dosing, including IFNg, TNFa, IL-2, IL-6 and IL-10. Transient increases in T-cell activation were observed by flow cytometry at 24-48 hours after ISB 1342 dosing at 1.0 / 4.0 μg/kg and above, based on increased expression of CD69 on peripheral blood CD4+ and CD8+ T-cell populations (Figure 1). Comparison of the available data from subjects treated at 2 / 8 μg/kg (Cohort 109) indicated reduced T-cell activation following SC compared to IV administration of ISB 1342. The efficacy signals observed in Cohorts 108 and 109 are consistent with the estimates of the lower boundary of the predicted efficacious dose range using a quantitative systems pharmacology (QSP) model based on preclinical data. Updated clinical data, including response rates and safety data, will be present at the meeting. Conclusions: Treatment with ISB 1342 was well tolerated at higher dose levels evaluated. Observed CRS events were moderate. No increased risk of infection has been observed . Updated safety, efficacy, biomarker and PK data will be presented.

Long-Term Follow-up and T Cell Characteristics of Patients with ASXL1-Mutated Relapsed or Refractory MDS or CMML Treated with Guadecitabine and Atezolizumab

Background Treatment of patients with myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML) after failure of a hypomethylating agent (HMA) remains an unmet need. We and others demonstrated a modest overall response rate (ORR) to the combination of immune checkpoint inhibition (ICI) and an HMA in such patients; a promising overall survival (OS) compared to historical data (5-6 months) was seen in some, but not all, studies. Our study and that of Daver et al. suggested that patients with ASXL1 mutations, in particular, may benefit from the addition of ICI. We conducted a Phase I/II clinical trial evaluating the addition of a programmed death ligand 1 (PD-L1) inhibitor, atezolizumab, to a subcutaneous prodrug of the HMA decitabine, guadecitabine. We evaluated the presence of common driver mutations in CD3- bone marrow mononuclear cells (BMMC) and CD3+ T lymphocytes. Among 27 tested patients, 12 had the ASXL1 mutations in the myeloid compartment and, of those, 5 patients' T cells carried the exact same mutation at a variant allele frequency (VAF) of >10%. Both patients with complete remission in the study (n=2) carried the mutation in both compartments (co-mutated) and median OS was significantly better for co-mutated patients (not reached) than for those with CD3- BMMC (myeloid) mutations (9.5 months) or wild type ASXL1 (16.5 months). Herein we report long-term follow-up of this cohort of patients, along with T cell programmed death-1 (PD-1) expression analyzed based on mutational status. Methods Details of the trial design and correlative plan are published. In the published study we used a VAF cutoff of 2% (1% for TP53) for BMMC and 10% for CD3+ T lymphocytes for the panel of 40 commonly mutated genes. In the current study we also evaluated patients with a T cell ASXL1 VAF of 5-10%. Patients have been followed for progression and survival every 6 months. Demographic and disease characteristics and outcome data were compared based on ASXL1 mutation status. Previously obtained T lymphocyte PD-1 expression data from samples obtained pre-treatment (PRE), during cycle 2 (EARLY) and after cycle 3 or 4 (LATE) were analyzed based on ASXL1 mutation status. Results Among 27 patients from 4 centers with DNA available for genetic analysis, 15 had no ASXL1 mutation ( ASXL1 WT), 5 had ASXL1 mutations restricted to the myeloid lineage, and 7 had ASXL1 mutations in both the myeloid and T-cell populations ( ASXL1 co-mutated). Two co-mutated patients had T cell VAFs of 5-10% and 5 had VAFs>10% (Figure 1). No baseline characteristic by ASXL1 mutational status was significantly associated with response to study treatment, though 6 of 7 co-mutated patients (vs 3/5 myeloid-only patients) had not responded to first-line HMA (refractory). With a median follow-up time of 64.9 months, median survival times were 48.9 months (15.1, 64.9+) for co-mutated patients with T cell VAF >10%, 34.9 months (8.5, NR) for co-mutated patients including those with T cell VAF of >5%, 16.4 months (3.3, 27.3) for ASXL1 WT patients, and 9.5 months (2.1, 35) for myeloid-only patients (Figure 2). This was statistically significant after adjusting for age and ECOG performance status at study entry, diagnosis, baseline bone marrow blast percentage, and number of prior chemotherapy regimens using the likelihood ratio test from multivariate analysis for overall survival (P = 0.008). Early treatment-induced upregulation of CD8 + T cell PD1 was observed in the ASXL1 WT, before returning to baseline levels. This upregulation was more pronounced in the ASXL1 co-mutated group beginning at a lower baseline and peaking at a higher MFI and was statistically significant (P = 0.0098). No significant treatment-induced change in PD-1 expression was noted in the myeloid-only subset. Discussion In patients with relapsed or refractory MDS or CMML, the presence of an ASXL1 mutation in both myeloid and T lymphocyte compartments, as compared to WT or myeloid-only mutations, was associated with a significantly longer median OS when treated with the combination of ICI and HMA. Significant upregulation of PD-1 was noted in T lymphocytes from both wild-type and co-mutated patients. The effect of mutant ASXL1 on T cell responsiveness to ICI deserves further investigation; patients with ASXL1-mutated HMA-refractory myeloid malignancies may benefit from ICI.

The Influence of TET2 and/or DNMT3A Knockout on the 5hmC Levels and Growth Characteristics of Modified Human Primary CD4 T Cells after Activation

Angioimmunoblastic T-cell lymphoma (AITL) has a unique immunohistological profile featuring malignant CD4+ T follicular helper cells (Tfh) inter-mixed with an extensive infiltrate of multiple lineages of immune cells. The AITL tumor microenvironment (TME) classically features small or large B cells (variably Epstein-Barr Virus (EBV) positive), reactive CD8+ T-cells, plasma cells and proliferative high endothelial venules (HEVs). Common genetic mutations have been identified within the malignant Tfh clone in AITL including epigenetic modifiers TET2, DNMT3A, and IDH2. DNMT3A mutations in AITL occur at a frequency of 20-38.5%and most of the DNMT3A mutations occur together with TET2 mutations. Tet2/ Dnmt3a double-knockout mice show synergistic effects on the expression of lineage-specifying transcription factor genes via epigenetic mechanisms. However, it is still not clear how DNMT3A mutations enhance the effect of TET2 mutations on promoting AITL development. To investigate how TET2 and DNMT3A mutations interact in AITL development, we first generated TET2 and/or DNMT3A knockout human primary CD4-T cells with CRISPR-Cas9 mediated genome editing. TET proteins could oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmc) in replication- independent DNA demethylation. Unmethylated cytosine base can be methylated by the DNA methylation machinery (DNMT3A/B) to form 5mC, which in turn provides the substrate for TET enzymes to produce 5hmC. TET and DNMT family members are thus needed to be dynamically modulate the epigenome to maintain cellular homeostasis or alter cellular function or development. We found that in response to CD3/CD28 stimulation, WT CD4-T cells exhibited division mediated dilutional reduction in 5 hmC which was later reconstitution. However, in TET2 or DKO cells, this process was compromised, showing delayed global 5hmc levels recovery (Fig. 1A). We then investigated whether the changes in methylation dynamics would alter cellular function. When using CD3/CD28 beads stimulation (10 days/cycle), we found that after two cycles of stimulation, these CD4-T cells with different gene editing started showing distinct behaviors with proliferation and apoptosis when exposed to CD3/CD28 beads again. The TET2/ DNMT3A double knockout (DKO) CD4-T cells showed more rapid response in cell division and higher proliferation rate than the other three groups (Fig. 1B). Compared with WT cells, consistently higher apoptotic cells were found in DNMT3A-KO group, but not with either TET2-KO or DKO cells (Fig. 1C). Interestingly, withdraw of beads at the end of the 10 days cycle, DKO cells kept higher baseline proliferation level than WT cells (Fig. 1D). We then used autologous or allogeneic EBV transformed B-cell for stimulation of the CD4 T-cells after one or two cycles of CD3/28 beads stimulation, and different response kinetics were observed among these prior activated CD4-T cells with TET2 and/or DNMT3A mutations. DKO cells showed more rapid response in cell division and elevated percentages of replicated cells to either autologous or allogeneic B-cells compared with WT cells (Fig. 1E). An interesting aspect of this B/T interaction is that, even the autologous B-cells could activate a large percentage of the T-cell population, particularly in the DKO cells, so it is not likely due to specific antigen recognition but rather a non-specific stimulation by the B-cells, probably via one or more co-stimulatory molecules (ICOSL, CD86 or 4-1BBL). This may be relevant to in vivo evolution of the mutated T-cells with B-cells/activated B-cells providing support.

DHODH Inhibitor Therapy for the Treatment of T-ALL Spares the Developing Thymus

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. Furthermore, T-ALL has not benefitted from immune-mediated therapies and continues to pose an ongoing treatment challenge with an unmet therapeutic need. DHODH has been highlighted as a potential therapeutic target in many hematologic and solid tumor malignancies including AML, neuroblastoma, glioblastoma, pancreatic cancer, and breast cancer. DHODH inhibition has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. We have confirmed this sensitivity in vitro as well as in vivo in three murine models of T-ALL, including an aggressive NOTCH driven model in syngeneic recipients and two human patient derived xenografts. Given the goal of making DHODHi therapy available to young children with relapsed/refractory T-ALL, we wished to understand the ramifications of nucleotide starvation on normal T-cells and other hematopoietic progenitors. To specifically address this, we turned our attention to the effects of DHODH inhibition on the developing thymus. This compartment would be of particular interest in pediatric patients, whose thymi have not yet finished developing. We studied juvenile mice (3 weeks) whose thymi are just reaching their peak size and growth rate. These mice were treated with the small molecule DHODHi brequinar (BRQ) 50 mg/kg every 72 hours, and were paired with age and gender-matched controls at each time point of analysis. A subset of mice were analyzed following two doses of BRQ, and thymus, bone marrow, spleen, and peripheral blood were analyzed for T-cell populations, both relative and absolute numbers. Another cohort was allowed to “recover” for 2 weeks following treatment before analysis, and a third cohort was allowed a four week recovery period. We discovered that initial treatment with brequinar significantly impacts T-cells across all stages of development ( Figure 1). This is expected, as developing T-cells are rapidly proliferative (often dividing as quickly as every 6 hours) and therefore highly dependent on their supply of nucleotides for DNA replication as well as transcription. However, after the two-week and the four-week recovery periods, these differences are no longer detectable, demonstrating the resilience of the developing thymus, and demonstrating that the effects of nucleotide starvation are temporary and reversible. These data are encouraging, as this may also suggest that the developing human thymus which is similarly rapidly proliferative in nature would also recover once nucleotide starvation was removed. Based on these data, we were interested in understanding the differential sensitivity of normal thymocytes as compared to malignant T-lymphoblasts to DHODHi. The resilience shown by normal thymocytes following treatment with BRQ may be due to their ability to tolerate periods of inhibition of de novo pyrimidine synthesis by preferentially relying on extracellular nucleotide salvage, whereas the sensitivity of malignant T-cells to DHODH may be due to their lack of ability to utilize the salvage pathway. We performed experiments to understand how cells balance de novo synthesis vs extracellular salvage. First, we performed metabolite flux tracing in Jurkat cells to directly measure nucleotide salvage. Here, we followed incorporation of isotopically labeled glutamine ( 15N) and uridine ( 13C) to determine the relative contributions of synthesis vs salvage, as de novo pyrimidine synthesis initiates from glutamine and aspartate while a cell can also use membrane nucleoside transporters (SLC28A and SLC29A family of proteins) to salvage extracellular uridine. These experiments were performed in the presence and absence of BRQ. Once BRQ is added and the cell's ability to synthesize pyrimidines is shut down, the cell must rely more on extracellular salvage as shown in Figure 2. A defective or less efficient process of extracellular salvage could help to explain this cell line's unusual dependence on de novo nucleotide synthesis. Overall, we hope that these findings will pave the way for additional preclinical studies and eventually clinical trials for pediatric patients with T-cell disease.

Prednisolone Intake Differentially Regulates IL12p40 and IL18 - Impact on Severe Chronic Gvhd, Relapse and Mortality

Background: Chronic graft-versus-host disease (cGVHD) is an immune-mediated complication after allogeneic stem cell transplantation (alloSCT). It is associated with graft-versus-leukemia effects and lower relapse rates, but in its severe form also with increased morbidity and mortality. The early interferon-gamma (IFNγ)-response marker CXCL9 is increased in mild and severe cGVHD and strongly correlates with IDO activity (1). Interleukin (IL)-18 is an innate, IFNγ inducing cytokine that associates with poor outcome of allografted patients (2), and with severe fibrosing cGVHD (1). The adaptive immunological arm of IFNγ induction relies on IL12 that is produced during cognate interactions between dendritic cells and T cells. IL12 kinetics is poorly characterized after alloSCT. We hypothesized that adaptive (IL12p40) and innate (IL18) immune mediators and their modulation by immunosuppressive drugs (ATG, prednisolone) impact cGVHD severity and outcome. Methods: Patients before alloSCT were included in an observational study allowing collection of clinical data and blood before and for one year after alloSCT. The training cohort included 350 patients transplanted before 2018 at our institution. The validation cohort consisted of 205 patients transplanted after 2018. Patients were eligible if they were not relapsed and did not receive donor leukocyte infusions (DLI) until d180, and did not develop refractory acute GVHD. Cytokines that belonged to the IFNγ pathway (IFNγ, CXCL9, CXCL10, IL12p40, IL12EBI3, IL17, IL18, IL23) or to innate inflammatory pathways (TNFa, IFNa, IL1b, activin, follistatin) were measured by R&D ELISAs. Cumulative (equivalent) prednisolone dosages between d0 and d100 after alloSCT were calculated from electronic patient records. Flow cytometry of PBMC of 100 patients on day+180/onset cGVHD were performed using 24-gate multicolor staining. Mild, severe fibrosing and severe gastro-intestinal (GI) cGVHD were diagnosed according to simplified NIH criteria (training cohort only). Overall survival, non-relapse mortality and time to relapse from time of blood sampling were calculated by multivariable Cox regression analyses. Results: Median time of blood sampling at onset of cGVHD (n=180) was day +168 (range 41-723). Control patients (n=169) were collected on day +180 (171-183). Mild cGVHD was recorded in 133 patients, severe fibrosing cGVHD in 39 patients, and isolated GI-cGVHD in 8 patients. Receiver-operator characteristic (ROC) curves showed that only CXCL9, IL18 and IL12p40 were significantly linked to severe cGVHD. IL18 was associated with severe fibrosing cGVHD, whereas CXCL9 and IL12p40 were correlated to any grade cGVHD. In multivariable Cox analyses including IL12p40, IL18, age, mismatch, recipient and donor gender, disease stage, ATG, MTX, and diagnosis, IL18 associated with poor OS (HR per log2 increase 1.81 (1.19-2.74, p=0.005), whereas IL12p40 associated with superior OS (HR 0.53 (0.35-0.81), p=0.003). This could be validated in the independent cohort. Time course analyses of serum levels of CXCL9, IL12p40 and IL18 revealed a strong increase of CXCL9 and IL12p40 toward onset of cGVHD, but no stimulation of IL18 levels. Cumulative prednisolone intake>100 mg until day+100 significantly reduced IL12p40 serum levels on d180/at onset of cGVHD, without influencing IL18 production (Figure 1). In contrast, ATG reduced both, IL18 and IL12p40 at day180/onset of cGVHD. This might explain the reduced incidence of severe fibrosing cGVHD in patients receiving ATG prophylaxis (days-3 to -1 before alloSCT, 4% vs. 23%, p<0.001, n=343), whereas no effect of early prednisolone intake was noticed (p=0.916, n=137). Multicolor flow cytometry showed that IL12p40, but not IL18 correlated with blood counts of T-lymphocyte populations, including CD4+, CD8+ and γδ-Tcells. IL12p40 and γδ-Tcells were associated with reduced relapse rates. In summary, CXCL9, IL12p40 and IL18 are associated with severe cGVHD. IL18 correlated with severe fibrosing cGVHD and increased mortality, whereas IL12p40 was associated with reduced NRM, reduced relapse risk, improved lymphocyte recovery and γδ-T-cells, and was affected by prednisolone intake but not ATG. Conclusion: These results may help to understand and potentially separate the GVL effects from mortality/morbidity that associate with cGVHD. 1)PMID34755129 2)PMID31680001

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC...

Diagnostic significance of blood lymphocyte activation markers in pre-eclampsia.

The adaptive and innate immune system is important in both initiating and preventing functional disorders during pregnancy, one of which is pre-eclampsia. The research aims to conduct the comparative quantification of selected subpopulations of peripheral blood immunoregulatory cells in pregnant women with pre-eclampsia in the third trimester. The marker receptors CD4, CD8, CD95, CD25, and CD27 and the marker antigen HLA-DR were considered. The screening was performed by flow cytometry with dual phenotyping using phycoerythrin- and fluorescein-isothiocyanate-labeled monoclonal antibodies. Data processing consisted in calculating a likelihood value to assess the statistical significance of the difference between the samples. A statistically significant decrease in the subpopulation titer of T and B lymphocytes with marker receptors CD4, CD8, and CD19 was found in pre-eclampsia patients. In the CD4 carrier T-lymphocyte population, there was an increased expression of the CD25/CD95 activation and apoptosis markers. In the CD8 T-killer population, a decreased representation of the CD27/CD25/CD95 markers of differentiation, activation, and apoptosis was deterministic. The expression pattern of the major histocompatibility complex antigen HLA-DR did not change significantly in normality and pathology. The titer of peripheral natural killer cells carrying the CD56 marker increased in patients with various degrees of disease severity, while the number of CD16 natural killer remained at the level of the control group. The research results suggest that a change in the ratio of the above receptors is a diagnostic indicator for pre-eclampsia.

CD8 effector T cells enhance teclistamab response in BCMA-exposed and -naïve multiple myeloma

AbstractTeclistamab, a B-cell maturation antigen (BCMA)- and CD3–targeting bispecific antibody, is an effective novel treatment for relapsed/refractory multiple myeloma (R/RMM), but efficacy in patients exposed to BCMA-directed therapies and mechanisms of resistance have yet to be fully delineated. We conducted a real-world retrospective study of commercial teclistamab, capturing both clinical outcomes and immune correlates of treatment response in a cohort of patients (n = 52) with advanced R/RMM. Teclistamab was highly effective with an overall response rate (ORR) of 64%, including an ORR of 50% for patients with prior anti-BCMA therapy. Pretreatment plasma cell BCMA expression levels had no bearing on response. However, comprehensive pretreatment immune profiling identified that effector CD8+ T-cell populations were associated with response to therapy and a regulatory T-cell population associated with nonresponse, indicating a contribution of immune status in outcomes with potential utility as a biomarker signature to guide patient management.

CD57 T cells associated with immunosenescence in adults living with HIV or AIDS.

Despite the advancement of human immunodeficiency virus (HIV)-related discoveries, new HIV infections still persist. With the advent of antiretroviral therapy, prognosis has migrated from acute to chronic HIV infection and inflammation, with the possibility of increased immune aging. We aimed to assess such immunosenescence by analysing CD27 and CD57 expression on the surface of T cells. This cross-sectional study was conducted between 2017 and 2018 on people living with HIV/AIDS (PLWHA) who attended an outpatient clinic of the Infectious Diseases Service of a university hospital and a geriatric reference service in Brazil. A standardized interview was conducted, and venous peripheral blood was collected for flow cytometry analysis. To assess immunosenescence, we compared CD27 and CD57 expression on the surface of T cells between adult and elderly individuals without HIV and adult PLWHA. All results for cells in terminal senescent stages in adult PLWHA more closely resembled those of elderly than adult participants without HIV (p > 0.05). The presence of CD27+ cells did not differ statistically among the three study groups when comparing immunological responders (IR) and immunological non-responders (INR); for the entire CD4+ T-cell population (including CD4 + CD8+ and CD4 + CD8- cells), the median count (25-75th) was higher in the INR (79.6%) than the IR (68.0%) group. HIV-infected individuals possessed a higher number of T lymphocytes with a molecular phenotype associated with immunosenescence, a lower proportion of T cells in the early stages of senescence (median 25-75th: 27.0%), and a higher proportion of T cells in the intermediate and final stages of senescence (median 25-75th: 16.1%) than adults without HIV (median 25-75th: 42.0% and 18.4%, respectively). Considering the higher number of senescent T lymphocytes, we observed in our PLWHA population-especially in the INR group (CD8CD57+ cells: 39.3% INR vs. 23.4% IR; CD4CD57+ cells: 44.0% INR vs. 27.7% IR)-may indicate a similar mortality risk phenotype from immune-preventable diseases.

A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.

A Cardiolipin from Muribaculum intestinale Induces Antigen-Specific Cytokine Responses.

An systematic phenotypic screen of the mouse gut microbiome for metabolites with an immunomodulatory effect identified Muribaculum intestinale as one of only two members with an oversized effect on T-cell populations. Here we report the identification and characterization of a lipid, MiCL-1, as the responsible metabolite. MiCL-1 is an 18:1-16:0 cardiolipin, whose close relatives are found on concave lipid surfaces of both mammals and bacteria. MiCL-1 was synthesized to confirm the structural analysis and functionally characterized in cell-based assays. It has a highly restrictive structure-activity profile, as its chain-switched analog fails to induce responses in any of our assays. MiCL-1 robustly induces the production of pro-inflammatory cytokines like TNF-α, IL-6, and IL-23, but has no detectable effect on the anti-inflammatory cytokine IL-10. As is the case with other recently discovered immunomodulatory lipids, MiCL-1 requires functional TLR2 and TLR1 but not TLR6 in cell-based assays.

Cancer therapeutic trispecific antibodies recruiting both T and natural killer cells to cancer cells.

T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fc‑fused TaKEs were designed, TaKE1‑Fc, TaKE2‑Fc and TaKE3‑Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1‑Fc was predicted to form a circular tetrabody‑like configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1‑Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of Tcell and NK cell populations was developed.

The menstrual cycle regulates migratory CD4 T-cell surveillance in the female reproductive tract via CCR5 signaling

Despite their importance for immunity against sexually transmitted infections, the composition of female reproductive tract (FRT) memory T-cell populations in response to changes within the local tissue environment under the regulation of the menstrual cycle remains poorly defined. Here, we show that in humans and pig-tailed macaques, the cycle determines distinct clusters of differentiation 4T-cell surveillance behaviors by subsets corresponding to migratory memory (TMM) and resident memory T cells. TMM displays tissue-itinerant trafficking characteristics, restricted distribution within the FRT microenvironment, and distinct effector responses to infection. Gene pathway analysis by RNA sequencing identified TMM-specific enrichment of genes involved in hormonal regulation and inflammatory responses. FRT T-cell subset fluctuations were discovered that synchronized to cycle-driven CCR5 signaling. Notably, oral administration of a CCR5 antagonist drug blocked TMM trafficking. Taken together, this study provides novel insights into the dynamic nature of FRT memory CD4T cells and identifies the menstrual cycle as a key regulator of immune surveillance at the site of STI pathogen exposure.

Hedgehog costimulation during ischemia-reperfusion injury potentiates cytokine and homing responses of CD4+ T cells.

Ischemia reperfusion injury (IRI) confers worsened outcomes and is an increasing clinical problem in solid organ transplantation. Previously, we identified a "PtchHi" T-cell subset that selectively received costimulatory signals from endothelial cell-derived Hedgehog (Hh) morphogens to mediate IRI-induced vascular inflammation. Here, we used multi-omics approaches and developed a humanized mouse model to resolve functional and migratory heterogeneity within the PtchHi population. Hh-mediated costimulation induced oligoclonal and polyclonal expansion of clones within the PtchHi population, and we visualized three distinct subsets within inflamed, IRI-treated human skin xenografts exhibiting polyfunctional cytokine responses. One of these PtchHi subsets displayed features resembling recently described T peripheral helper cells, including elaboration of IFN-y and IL-21, expression of ICOS and PD-1, and upregulation of positioning molecules conferring recruitment and retention within peripheral but not lymphoid tissues. PtchHi T cells selectively homed to IRI-treated human skin xenografts to cause accelerated allograft loss, and Hh signaling was sufficient for this process to occur. Our studies define functional heterogeneity among a PtchHi T-cell population implicated in IRI.

Dysfunctional states of unconventional T-cell subsets in cancer.

Unconventional T cells represent a promising therapeutic agent to overcome the current limitations of immunotherapies due to their universal T-cell receptors, ability to respond directly to cytokine stimulation, and capacity to recruit and modulate conventional immune cells in the tumor microenvironment. Like conventional T cells, unconventional T cells can enter a dysfunctional state, and the functional differences associated with this state may provide insight into the discrepancies observed in their role in antitumor immunity in various cancers. The exhaustive signature of unconventional T cells differs from conventional αβ T cells, and understanding the differences in the mechanisms underlying exhaustive differentiation in these cell types may aid in the discovery of new treatments to improve sustained antitumor responses. Ongoing clinical trials investigating therapies that leverage unconventional T-cell populations have shown success in treating hematologic malignancies and reducing the immunosuppressive tumor environment. However, several hurdles remain to extend these promising results into solid tumors. Here we discuss the current knowledge on unconventional T-cell function/dysfunction and consider how the incorporation of therapies that modulate unconventional T-cell exhaustion may aid in overcoming the current limitations of immunotherapy. Additionally, we discuss how components of the tumor microenvironment alter the functions of unconventional T cells and how these changes can affect tumor infiltration by lymphocytes and alter conventional T-cell responses.

Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.

Tumor-targeted delivery of lnc antisense RNA against RCAS1 by live-attenuated tryptophan-auxotrophic Salmonella inhibited 4T1 breast tumors and metastasis in mice

Emerging chemo- and radiotherapy resistance exacerbated thecancer risk and necessitated novel treatment strategies. Although RNA therapeutics against pro-oncogenic genes are highly effective, tumor-specific delivery remains a barrier to the implementation of this valuable tool. In this study, we report a tryptophan-auxotrophic Salmonella typhimurium strain as an onco-therapeutic delivery system with tumor-targeting ability using 4T1 mice breast-cancer model. The receptor-binding cancer antigen expressed on SiSo cell (RCAS1) is a cancer-specific protein that induces the apoptosis of peripheral lymphocytes and confers tumor immune evasion. We designed a long non-coding antisense-RNA against RCAS1 (asRCAS1) and delivered by Salmonella using a non-antibiotic, auxotrophic-selective, eukaryotic expression plasmid, pJHL204. After invivo tumor-to-tumor passaging, the JOL2888 (ΔtrpA, ΔtrpE, Δasd+ asRCAS1) strain exhibited high sustainability in tumors, but did not last in healthy organs, thereby demonstrating tumor specificity and safety. RCAS1 inhibition in the tumor was confirmed by western blotting and qPCR. In mice, JOL2888 treatment reduced tumor-associated macrophages, improved the Tcell population, elicited cell-mediated immunity, and suppressed cancer-promoting genes. Consequently, the JOL2888 treatment significantly decreased the tumor volume by 80%, decreased splenomegaly by 30%, and completely arrested lung metastasis. These findings highlight the intrinsic tumor-targeting ability of tryptophan-auxotrophic Salmonella for delivering onco-therapeutic macromolecules.

Loss of αβ but not γδ Tcells in chickens causes a severe phenotype.

The availability of genetically modified mice has facilitated the study of mammalian Tcells. No model has yet been developed to study these cells in chickens, an important livestock species with a high availability of γδ Tcells. To investigate the role of γδ and αβ Tcell populations in birds, we generated chickens lacking these Tcell populations. This was achieved by genomic deletion of the constant region of the Tcell receptor γ or β chain, leading to a complete loss of either γδ or αβ Tcells. Our results show that a deletion of αβ Tcells but not γδ Tcells resulted in a severe phenotype in KO chickens. The αβ Tcell KO chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αβ Tcell KO chickens revealed a significant increase in monocytes and expectedly the absence of CD4+ Tcells including FoxP3+ regulatory Tcells. Surprisingly there was no increase of γδ Tcells. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of Tcell knockouts in chickens and provide new insights into their function in vertebrates.