Low CD20 Expression Research Articles

CD146 Molecule Expression in B Cells Acute Lymphoblastic Leukemia (B-ALLs): A Flow-Cytometric Marker for an Accurate Diagnostic Workup.

B-lineage acute lymphoblastic leukemias (B-ALL) harboring the t(9;22)(q34;q11)/BCR::ABL1 rearrangement represent a category with previously dismal prognosis whose management and outcome dramatically changed thanks to the use of tyrosine kinase inhibitors (TKIs) usage and more recently full chemo-free approaches. The prompt identification of these cases represents an important clinical need. We sought to identify an optimized cytofluorimetric diagnostic panel to predict the presence of Philadelphia chromosome (Ph) in B-ALL cases by the introduction of CD146 in our multiparametric flow cytometry (MFC) panels. We prospectively evaluated a total of 245 cases of newly diagnosed B-ALLs with a CD146 positivity threshold >10% referred to the Division of Hematology of 'Sapienza' University of Rome. We compared the results of CD146 expression percentage and its mean fluorescence intensity (MFI) between Ph+ ALLs, Ph-like ALLs, and molecularly negative ALLs. Seventy-nine of the 245 B-ALL cases (32%) did not present mutations at molecular testing, with 144/245 (59%) resulting in Ph+ ALL and 19/245 (8%) Ph-like ALLs. Comparing the 3 groups, we found that Ph+ B-ALLs were characterized by higher expression percentage of myeloid markers such as CD13, CD33, and CD66c and low expression of CD38; Ph+ B-ALL showed a higher CD146 expression percentage and MFI when compared with both molecular negative B-ALL and Ph-like ALLs; neither the mean percentage of CD146 expression neither CD146 MFI were statically different between molecular negative B-ALL and Ph-like ALLs. Our data demonstrate the association between CD146 expression and Ph+ ALLs. CD146, along with myeloid markers, may help to identify a distinctive immunophenotypic pattern, useful for rapid identification in the diagnostic routine of this subtype of B-ALLs that benefits from a specific therapeutic approach.

Immunohistochemical expression of CD44 in invasive breast carcinoma

Background: In India, breast cancer forms the most common malignancy, accounting for 28.2% of all female cancers. Breast cancer consists of phenotypically diverse group of diseases. Due to different relapse abilities, doubling times, and different infiltrative capacities, it is important to identify potential biomarkers that could be used to screen high-risk patient and predict breast cancer prognosis in conjunction with classical pathological parameters. Aims and Objectives: The present study was conducted to evaluate immunohistochemical expression of CD44 in breast carcinoma and to correlate CD44 expression with other clinicopathological parameters and molecular classification of carcinoma breast. Materials and Methods: The present study was a prospective descriptive study conducted on 100 cases of carcinoma breast diagnosed on modified radical mastectomy specimens that were submitted to the Department of Pathology, Pt. B. D. Sharma, PGIMS, Rohtak, Haryana, over the period of 2 years. Results: Out of 100, 64 cases (64%) were CD44 positive. Out of these 64 cases, 9 (9%) cases were 1+, 34 (34%) cases were 2+, and 21 (21%) cases showed 3+ positive expression of CD44 in tumor cells. Loss of CD44 expression was seen in 36 (36%) cases. A statistically significant association of CD44 was seen with multifocality of the tumor, negative estrogen receptor/progesterone receptor (ER/PR) expression, and molecular subtypes. No statistically significant association of CD44 was seen with age, side of tumor, lymph node metastasis, lymphovascular invasion, ductal carcinoma in situ and Ki67, and HER2neu expression. Conclusion: A high CD44 expression in breast carcinoma correlates with aggressive tumor characteristics such as multifocality, negative ER/PR expression, and belonging to the basal subtype which is associated with poor prognosis. Conversely, low CD44 expression is linked to Luminal A and Luminal B subtypes which have good prognosis. These indicate the role of CD44 as a prognostic biomarker in the carcinoma breast.

A dual-targeting approach with anti-IL10R CAR-T cells engineered to release anti-CD33 bispecific antibody in enhancing killing effect on acute myeloid leukemia cells.

Immunotherapies, including chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs), encounter several challenges in the management of acute myeloid leukemia (AML), including limited persistence of these treatments, antigen loss and resistance of leukemia stem cells (LSCs) to therapy. Here, we proposed a novel dual-targeting approach utilizing engineered anti-IL10R CAR-T cells to secrete bispecific antibodies targeting CD33. This innovative strategy, rooted in our previous research which established a connection between IL-10 and the stemness of AML cells, designed to improve targeting efficiency and eradicate both LSCs and AML blasts. We first demonstrated the superior efficacy of this synergistic approach in eliminating AML cell lines and primary cells expressing different levels of the target antigens, even in cases of low CD33 or IL10R expression. Furthermore, the IL10R CAR-T cells that secret anti-CD33 bsAbs (CAR.BsAb-T), exhibited an enhanced activation and induction of cytotoxicity not only in IL10R CAR-T cells but also in bystander T cells, thereby more effectively targeting CD33-positive tumor cells. Our in vivo experiments provided additional evidence that CAR.BsAb-T cells could efficiently redirect T cells, reduce tumor burden, and demonstrate no significant toxicity. Additionally, delivering bsAbs locally to the tumor sites through this strategy helps mitigate the pharmacokinetic challenges typically associated with the rapid clearance of prototypical bsAbs. Overall, the engineering of a single-vector targeting IL10R CAR, which subsequently secretes CD33-targeted bsAb, addresses the issue of immune escape due to the heterogeneous expression of IL10R and CD33, and represents a promising progress in AML therapy aimed at improving treatment outcomes.

Surface-engineered erythrocyte membrane-camouflage fluorescent bioprobe for precision ovarian cancer surgery.

Fluorescence imaging-guided surgery has been used in oncology. However, for tiny tumors, the current imaging probes are still difficult to achieve high-contrast imaging, leading to incomplete resection. In this study, we achieved precise surgical resection of tiny metastatic cancers by constructing an engineering erythrocyte membrane-camouflaged bioprobe (AR-M@HMSN@P). AR-M@HMSN@P combined the properties of aggregation-induced emission luminogens (AIEgens) named PF3-PPh3 (P), with functional erythrocyte membrane modified by a modular peptide (AR). Interestingly, AR was composed of an asymmetric tripodal pentapeptide scaffold (GGKGG) with three appended modulars: KPSSPPEE (A6) peptide, RRRR (R4) peptide and cholesterol. To verify the specificity of the probe in vitro, SKOV3 cells with overexpression of CD44 were used as the positive group, and HLF cells with low expression of CD44 were devoted as the control group. The AR-M@HMSN@P fluorescence imaging was utilized to provide surgical guidance for the removal of micro-metastatic lesions. In vivo, the clearance of AR-M@HMSN@P by the immune system was reduced due to the natural properties inherited from erythrocytes. Meanwhile, the A6 peptide on AR-M@HMSN@P was able to specifically target CD44 on ovarian cancer cells, and the electrostatic attraction between the R4 peptide and the cell membrane enhanced the firmness of this targeting. Benefiting from these multiple effects, AR-M@HMSN@P achieved ultra-precise tumor imaging with a signal-to-noise ratio (SNR) of 15.2, making it possible to surgical resection of tumors < 1mm by imaging guidance. We have successfully designed an engineered fluorescent imaging bioprobe (AR-M@HMSN@P), which can target CD44-overexpressing ovarian cancers for precise imaging and guide the resection of minor tumors. Notably, this work holds significant promise for developing biomimetic probes for clinical imaging-guided precision cancer surgery by exploiting their externally specified functional modifications.

Preclinical assessment of IPH6501, a first-in-class IL2v-armed tetraspecific NK cell engager directed against CD20 for R/R B-NHL, in comparison with a CD20-targeting T cell engager.

7030 Background: The therapeutic landscape for relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL) is evolving to include targeted T-cell based immunotherapies, including CD19-targeted CAR-T and CD3xCD20 T-cell engaging (TCE) bispecific antibodies. Yet, there remains an unmet medical need for patients who are refractory to, or ineligible for these treatments. Leveraging natural killer (NK) cells emerges as a promising strategy in hematological malignancies, as shown in a Phase 1 study with IPH6101/SAR’579 in R/R AML (Stein, ASCO 2023; Bajel, ASH 2023). Methods: We developed IPH6501, a tetraspecific antibody-based NK cell engager that simultaneously targets on NK cells the CD16a and NKp46 receptors, the IL-2 receptor with an engineered IL-2 variant (IL2v) and on B-NHL cells the CD20 antigen. This approach boosts NK cell activation and proliferation, cytotoxicity against tumor cells, and cytokine production. The IL-2 variant is designed with mutations that prevent binding to CD25 (IL-2Rα), limiting Treg activation and potential IL-2-related side effects. IPH6501 has been evaluated in mouse, non-human primate, and human-derived models, including cells from R/R B-NHL patients. Results: In vivo studies in non-human primates and tumor mouse models revealed that IPH6501, at well-tolerated doses, significantly boosted peripheral NK cell proliferation, drove their accumulation at tumor locations, and effectively eradicated CD20+ cells in blood and tissues. In human derived-models, IPH6501 significantly induced NK cell proliferation and cytotoxicity. Notably, IPH6501 showed potent activity against a range of B-NHL cell lines, including those with low CD20 expression. In addition, IPH6501 upregulated the expression of activating receptors on NK cells, such as NKG2D, essential for the recognition and killing of malignant cells, thereby introducing another mode of action enabling the elimination of CD20-negative tumor cells. In a comparative analysis with a CD3xCD20 TCE, IPH6501 induced lower cytokine secretion, suggesting a potentially safer profile. Additionally, IPH6501 showed higher killing efficacy compared to the TCE in samples from healthy individuals as well as R/R B-NHL patients, underscoring its therapeutic potential. Conclusions: The preclinical demonstrations of IPH6501 activities across various in vivo models and its effectiveness in ex vivo assays using cells from R/R B-NHL patients provide compelling evidence of its therapeutic potential and tolerability. IPH6501 is emerging as a promising new candidate within the treatment landscape for R/R B-NHL and is currently being investigated in a global, first-in-human phase 1/2 study (NCT 06088654).

Hyaluronic acid-coated liposomes for enhanced in vivo efficacy of neogambogic acid via active tumor cell targeting and prolonged systemic exposure

Neogambogic acid (NGA), which possesses a variety of anticancer activities, is visualized as an anticancer bioactive ingredient. However, the huge vascular stimulation, poor aqueous solubility, and short half-life restricted its clinical use. In this work, an effective nanocarrier was explored to reduce toxicity and enhance the tumor-targeted delivery. Two liposomal formulations, neogambogic acid liposomes (NGA-L), and hyaluronic acid-coated neogambogic acid liposomes (HA-NGA-L) were prepared and characterized with high encapsulation efficiency, slow pattern of drug release, narrow size distribution and higher stability. The cytotoxicity and cellular uptake of HA-NGA-L were higher than those of NGA-L in MDA-MB-231 cells (high CD44 expression), while no obvious differences in MCF-7 cells with (low CD44 expression), suggesting the CD44-mediated cellular internalization of hyaluronic acid-modified liposomes enhanced the cytotoxicity. Mechanistically, elevation of Bax and caspase-3 as well as downregulation of Bcl-2 led to cell apoptosis. Besides, the vascular stimulation and the hemolysis test indicated good safety of HA-NGA-L. In addition, HA-NGA-L was the effective nanocarrier to repress tumor proliferation in MDA-MB-231 tumor xenograft mouse through CD44 mediated active targeting without any obvious histopathological abnormities on major organs. Immunohistochemistry analysis revealed the enhanced elevation of Bax and caspase-3, and reduced expression of Bcl-2 contribute to apoptosis in tumors. Meanwhile, HA-NGA-L increased the AUC and t1/2 by 5.34-fold and 3.94-fold, respectively. In summary, the present study shows that HA-NGA-L may be safe and effective for the tumor-targeted delivery of neogambogic acid.

The Expression Levels of CD20 as a Prognostic Value in Feline B-Cell Nasal Lymphoma: A Pilot Study.

The effect of the semi-quantitative expression of CD20 in the prognosis of feline nasal lymphoma has not been described. This study investigated the prognostic significance of CD20 expression, clinicopathological characterization, and treatment outcomes in cats with nasal lymphoma. Clinical data from cats diagnosed with nasal lymphoma were retrospectively collected, including signalment, clinical signs, clinicopathological variables, treatment outcomes, and survival times. Using ImageJ software, CD20 expression was semi-quantitatively measured based on the proportion of CD20-positive areas. Correlations between laboratory findings, immunohistochemical expressions, and survival outcomes were investigated. All cats included in the study exhibited the B-cell immunophenotype. During treatment, a reduction in PCV was noted in the cats at the second and sixth weeks (p = 0.01 and p = 0.01, respectively). The cats with low CD20 expression exhibited a significantly shorter MST (91 days; 95% CI, 41-141) than those with high CD20 expression (MST, 214 days; 95% CI, 76-351) (p = 0.01). Stage T1 cats displayed a higher MST (143 days; 95% CI, 144-172) than those in other stages > T1 (120 days, 95% CI, 71-169 days) (p = 0.04). Anemia, a common adverse effect in feline nasal lymphoma, did not impact MST. T1 clinical staging and high CD20 expression showed a trend for better MST.

Abstract 5171: CD39, CD73 and CD38 as potential biomarkers for monitoring the response to mogamulizumab in Sézary syndrome

Abstract We demonstrated aberrant expression of CD39 or CD73 and low expression of CD38 in circulating and skin-homing CD4+ T cells in patients with Sézary syndrome (SS), an erythrodermic cutaneous T-cell lymphoma (CTCL) with a leukemic component. The anti-CCR4 monoclonal antibody Mogamulizumab (Moga) has been shown to increase progression-free survival in patients with advanced SS. Here, we longitudinally explored the modulation of the expression of CD39, CD73 and CD38 in circulating CD4+ T cells from SS patients under treatment with Moga. Eight patients with relapsed/refractory SS were included in the study and prospectively followed up for two years (median duration of treatment 9,5 months, range 5-28 months). Seven out of eight (87,5%) patients showed early complete response in the blood and partial (5/7, 71,4%) or near complete response (2/7, 28,6%) in the skin. One patient (12,5%) showed no blood or skin response to the treatment. Flow cytometry analysis highlighted that the clinical response was accompanied by a rapid and remarkable increase in CD38 expression in the residual circulating CD4+ T cells, as well as in CD8+ T cells. Moreover, in six patients carrying the homozygous G/G or heterozygous A/G ENTPD1 SNP rs10748643 genotypes, permissive to CD39 overexpression in T cells, the increased CD38 expression was paralleled by rapid reduction/loss of CD39. Notably, in one patient carrying the A/A genotype, characterized by low CD39 and high CD73 expression at the baseline, response to treatment was characterized by reduction of CD73 expression. The only one patient with A/A genotype who didn’t respond to Moga showed no modulation of CD38 or CD39 expression, while CD73 expression increased. Noteworthy, Moga increases the expression of CD38 in responding patients, and influences the expression of CD39 only in patients with the permissive CD39 genotypes (G/G; A/G), but not in those with the A/A genotype. Two patients showing initial response to Moga experienced skin progression during treatment, which was associated with a significant increase of CD39 and simultaneous decrease of CD38 in residual circulating CD4+ T cells, although there was no evidence of blood relapse. These preliminary results suggest that partial or complete responses to Moga is paralleled by reduced CD39 and/or CD73 expression and increased CD38 expression. Instead, an opposite modulation of these markers is associated with disease progression hinting at the potential clinical utility of CD38, CD39 and CD73 as markers to monitor patients' response to Moga therapy. Citation Format: Yuliya Yakymiv, Sara Marchisio, Erika Ortolan, Verdiana Pullano, Chiara Leso, Rebecca Senetta, Lorenzo Marega, Gabriele Roccuzzo, Pietro Quaglino, Ada Funaro. CD39, CD73 and CD38 as potential biomarkers for monitoring the response to mogamulizumab in Sézary syndrome [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 5171.

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [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 1237.

Abstract 1238: ISB 2001, a BCMA and CD38 dual targeting T cell engager, demonstrates superior cytotoxicity relative to teclistamab in the samples of patient relapsing from CD38 and BCMA targeted immunotherapies

Abstract Immune therapies targeting single tumor associated antigens (TAA) demonstrated efficacy against multiple myeloma (MM). However, durable responses are still limited1 potentially due to expansion of clones with low target expression2 We have previously demonstrated that simultaneous targeting of BCMA and CD38 on MM tumor cells with heterogenous expression of these antigens using dual targeting ISB 2001, enables superior killing relative to mono-targeting T cell engagers (TCE) 3. Here we present a further characterization of a dual targeting TCE utilizing bone marrow aspirates from multiple myeloma patients. In relapsed/refractory (r/r) patients, which received CD38 targeted therapy, daratumumab cytotoxicity was substantially reduced due to low CD38 expression. Teclistamab is considered the next line of treatment in such patients. However, ISB 2001 consistently demonstrated increased cytotoxicity compared to teclistamab in both newly diagnosed patient and r/r patient samples. Remarkably, ISB 2001 also induced stronger cytotoxic response in one patient relapsing after BCMA targeted therapy, suggesting that the dual targeting ISB 2001 TCE can overcome the escape mechanisms. ISB 2001 was also compared to the combination of teclistamab and daratumumab in cytotoxic assay using healthy donors. ISB 2001 shows superiority to both teclistamab and daratumumab as single agents, as well as when they were used in combination. Additionally, ISB 2001 was compared against a combination of daratumumab and teclistamab in a humanized mouse model of multiple myeloma with a low expression of both CD38 and BCMA, mimicking potential tumor escaping clones. ISB 2001 induced complete eradication of the tumors in almost all animals, whereas the combination only showed partial tumor protection, underscoring that dual targeting by a TCE is superior to two therapeutic agents individually targeting the same antigens. Based on the promising preclinical in vitro, ex vivo and in vivo data, we have advanced ISB 2001 into clinical studies. For calculating the first-in-human (FIH) dose, we have developed a quantitative systems pharmacology (QSP) model. The calculated FIH dose was 50-100 fold higher than that generated by a traditional minimum anticipated biological effect level calculation. Therefore, using this approach will substantially reduce patient exposure to sub-efficacious doses of ISB 2001. This plan was accepted by the HREC in Australia and the FDA in the US to initiate a Phase 1 FIH study of ISB 2001 for the treatment of relapsed/refractory multiple myeloma (NCT05862012)4. 1. Munshi, N. C. et al. 384, 705-716 (2021). 2. Nijhof, I. S. et al. Blood 128, 12 (2016). 3. (https://doi.org/10.1182/blood-2022-159353) 4. Abstract# 3396, ASH 2023, Hanlon Sia at al. Citation Format: Laura Carretero-Iglesia, Maria Pihlgren, Jeremy Berret, Adam Drake, Daniela Pais, Cyrille Dreyfus, Vinu Menon, Thomas Matthes, Claire Edwards, James Edwards, Catherine Pellat-Deceunynck, Philippe Moreau, Cyrille Touzeau, Tomomi Matsuura, Piet van der Graaf, Lida Pacaud, Cyril Konto, Eugene Zhukovsky, Mario Perro. ISB 2001, a BCMA and CD38 dual targeting T cell engager, demonstrates superior cytotoxicity relative to teclistamab in the samples of patient relapsing from CD38 and BCMA targeted immunotherapies [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 1238.

Abstract 1223: KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma

Abstract Daratumumab (Dara), a first-in-class humanized monoclonal antibody targeting CD38, demonstrates notable efficacy in both newly diagnosed and relapsed/refractory multiple myeloma (MM) when administrated alone or in combination with other agents. However, the development of Dara resistance and subsequent relapse is common.To identify the tumor-intrinsic genes involved in resistance to NK-mediated cytotoxicity in the presence of Dara in MM cells, we conducted an in vitro genome-wide CRISPR-Cas9 knockout (KO) screen and found 433 genes exhibiting a positive correlation with MM cytotoxicity elicited by Dara and primary NK cells. Remarkably, 16 of these genes demonstrated a consistent correlation with cytolytic activity across the majority of the 36 cancer types in the TCGA database.The efficacy of Dara is closely linked to the expression of CD38, with nonresponders exhibiting low CD38 expression. Therefore, we conducted another CRISPR screen to identify the genes whose KO resulted in diminished CD38 expression. The genes that overlapped in these two screens are implicated in regulating both Dara-mediated ADCC and CD38 expression. Our focus turned to KDM6A among the overlapping genes. We validated that KDM6A KO significantly decreased CD38 expression and that reintroducing KDM6A into KDM6A KO cells restored the expression of CD38 and Dara-induced cytotoxicity.KDM6A demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. Our ChIP-seq data unveiled that KDM6A-KO cells exhibited an increased H3K27me3 level at the CD38 promoter area than control cells. Remarkably, this pattern was consistent with Dara-resistant cell lines, which displayed reduced CD38 expression alongside elevated levels of H3K27me3 at the CD38 promoter. Interestingly, we found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment, implying the existence of an additional mechanism. We performed RNA-seq in KDM6A KO cells and found CD48, an NK-activating ligand, was significantly repressed. Overexpression of CD48 in KDM6A KO cells resulted in a substantial increase in the secretion of Granzyme B and Perforin by NK cells and restored the activity of NK cells and re-sensitized KDM6A KO cells to Dara treatment. These data suggest that KDM6A mediates Dara-mediated ADCC sensitivity through the dual regulation of CD38 and CD48. KDM6A acts to oppose the EZH2/PRC2 complex by demethylating H3K27me3, and we found that Tazemetostat (Taze), an FDA-approved EZH2 inhibitor, increased the expression of CD38 and CD48, especially in KDM6A-KO cells. Importantly, Taze also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. In summary, our findings illuminate the dual role of KDM6A in enhancing the efficacy of Dara in MM. Imitating KDM6A function using an FDA-approved inhibitor holds promise in overcoming Dara resistance and improving patient outcomes in MM. Citation Format: Jiye Liu, Lijie Xing, Jiang Li, Kenneth Wen, Ning Liu, Yuntong Liu, Annamaria Gulla, Nikhil Munshi, Teru Hideshima, Kenneth Carl Anderson. KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma [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 1223.

Outcomes Associated With ABCG2 and CD133 Expression in Patients With Gastric Cancer After Surgical Resection

Objectives: Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) and CD133 are recognized stem cell markers of gastric cancer. Extensive research has examined the significance of these markers in different types of cancers and their impact on prognoses, linking them to unfavorable clinical outcomes in various tumors. However, the prognostic value of these markers for gastric cancer remains unclear. We investigated the expression of ABCG2 and CD133 and their relationship with clinical outcomes in gastric cancer.Methods: ABCG2 and CD133 expression levels were analyzed, using immunohistochemistry and tissue microarrays, in tumor samples from 459 patients who underwent surgical resections due to gastric cancer. ABCG2 and CD133 expression levels were defined by intensity and dichotomized as medians. The associations among the expression levels of these markers, disease severity, and patient survival were also determined.Results: In the 411 patients for whom we analyzed the expression levels of these markers, 74.9% and 80.5% were found to have high levels of ABCG2 and CD133, respectively. High expression levels of ABCG2 and CD133 were more commonly observed in well-differentiated (&lt;i&gt;p&lt;/i&gt;&lt;0.001 and &lt;i&gt;p&lt;/i&gt;=0.004, respectively) and intestinal lesions (&lt;i&gt;p&lt;/i&gt;&lt;0.001 and &lt;i&gt;p&lt;/i&gt;=0.002, respectively). High ABCG2 expression correlated with improved survival outcomes, whereas high CD133 expression was associated with poorer outcomes. Cox regression analysis confirmed that stage, high ABCG2 (overall survival [OS]: hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.41–0.91; &lt;i&gt;p&lt;/i&gt;=0.015; recurrencefree survival [RFS]: HR, 0.55; 95% CI, 0.34–0.88; &lt;i&gt;p&lt;/i&gt;=0.012), and CD133 expression (OS: HR, 1.59; 95% CI, 1.00–2.51; &lt;i&gt;p&lt;/i&gt;=0.049; RFS: HR, 2.29; 95% CI, 1.21–4.34; &lt;i&gt;p&lt;/i&gt;=0.011) were predictors of survival. A subgroup analysis indicated that ABCG2 expression was also associated with an improved RFS rate in patients who received systemic chemotherapy.Conclusions: High ABCG2 expression and low CD133 expression in tumors correlated with improved survival outcomes in post-resection patients with gastric cancer, suggesting their potential utility as prognostic biomarkers.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Differential CD38 Expression on Daratumumab-Naïve Vs. Resistant Multiple Myeloma - a Direct Stochastic Optical Reconstruction Microscopy ( dSTORM) Analysis in 53 Patients

Background: The anti-CD38 antibody Daratumumab (Dara) has become the new standard of care for the treatment of newly-diagnosed and relapsed/ refractory multiple myeloma (MM). At the same time, Dara resistance remains poorly understood. Potential resistance mechanisms have been proposed and range from intrinsic downregulation in CD38 dim MM cells to genetic alterations and splicing variants which may affect CD38 epitope expression (Adamia, ASH 2022). Here, we used super-resolution microscopy for high-precision quantification of CD38 in MM and piloted this approach in a large cohort of 53 MM patients with vs. without Dara resistance. Methods: Direct stochastic optical reconstruction microscopy ( dSTORM) is a novel imaging modality based on photoswitching of certain fluorophores coupled to antibodies for molecular representation of surface molecules at unprecedented levels of resolution (&amp;lt;20 nM). To correlate CD38 expression data with clinical outcome, we divided our cohort based on treatment status into two groups: Dara-naïve (n = 31) vs. Dara-resistant (n = 22) patients. Imaging data was complemented by phenotypic assessment using a well-established multi-epitope anti-CD38 antibody (Cytognos), as per standard-of-care. Results: Mean CD38 receptor density as measured with Dara via dSTORM was significantly higher in Dara-naïve (of 48.81 ± 29.65 receptors/µm²) vs. Dara-resistant patients (15.52 ± 3.9 receptors/µm², p = 0.016). This finding was corroborated by multi-epitope phenotypic analysis which revealed mean CD38 receptor expression levels of 32.09 ± 13.14 receptors/µm² in Dara-naïve vs. 21.65 ± 17.73 receptors/µm² in Dara-resistant patients ( p = 0.05). Lower CD38 expression via dSTORM vs. multi-epitope anti-CD38 staining was consistently observed in our cohort and may reflect ongoing blockage of the receptor binding site by Dara in cases with shorter time to last Dara application (median 53 days, range 1-189 days). As there was no correlation with the interval between Dara application and sampling however, splice-site mutations affecting CD38 receptor accessibility as proposed by Adamia et al. may depict a more relevant mechanism of resistance in our Dara-resistant cohort. In a next step, we therefore investigated alternative epitope binding in Dara-resistant patients. To this end, we investigated antibody binding by dSTORM imaging for isatuximab (Isa), an alternative CD38-directed monoclonal antibody that binds to a location in the C-terminal portion of the CD38 extracellular domain which is entirely different from the Dara epitope. Interestingly, there was a trend for increased detection rate with Isa for mean CD38 receptor expression in Dara-resistant patients (22.2 ± 4.6 receptors/µm²) vs. Dara (15.5 ± 3.9 receptors/µm², p = 0.1) and on par with CD38 receptor expression. No data was available to assess Dara binding in Isa pre-exposed patients. Conclusion: Innovative microscopy using dSTORM allows for high-resolution epitope quantification at unprecedented granularity. While this finding enables visual proof of differential epitope binding of Dara vs. Isa on CD38 in a clinically feasible setting, correlation studies to determine the clinical relevance of this observation remain ongoing.

Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16/NKG2D Immune Modulating TriNKET ® BMS-986357 (CC-96191)

CD33 is widely expressed by myeloid cells and is a validated drug target in acute myeloid leukemia (AML), as shown by the benefit of some patients from the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO). However, GO is often ineffective and toxic, prompting interest in more potent CD33-directed therapies. Success of T cell-engaging bispecific antibodies in lymphoid malignancies has raised enthusiasm for similar agents targeting CD33, but toxicities from activated T cells cause significant challenges for their use and provide a strong rationale to explore drugs engaging natural killer (NK) cells. Here, we investigated BMS-986357, a TriNKET ® that activates NK cells via CD16 and NKG2D receptors while targeting CD33. This format was chosen because simultaneous engagement of multiple activating NK cell receptors yields greater cytotoxic activity against neoplastic cells than engagement via a single activating receptor. Surface plasmon resonance analyses showed BMS-986357 simultaneously binds CD33, NKG2D, and CD16a, and co-engagement of NKG2D and CD16a increases the avidity. In the presence of primary human NK cells from healthy donors or a human NK cell line, the TriNKET ® demonstrated broad cytolytic anti-leukemia activity in a strictly CD33-dependent manner using a panel of CD33-expressing or CD33-deficient/deleted acute leukemia cells. Consistent with this, removing the CD33 binding domain rendered BMS-986357 ineffective against CD33+ AML cells. Drug-induced cytotoxicity was greater with BMS-986357 than with monoclonal CD33 antibodies, including lintuzumab and an afucosylated antibody with enhanced NK function, demonstrating the benefit of the TriNKET ® format. Compared to BMS-986357, substantially less cytotoxicity was observed with an Fc-mutant variant of BMS-986357 that abrogates binding to CD16a or an NKG2D dead-arm TriNKET ®, and cytolytic effects were reduced with a CD16-deficient NK cell line vs. a CD16-expressing subline, indicating maximal efficacy of BMS-986357 required co-engagement of CD16a and NKG2D. Using sublines of OCI-AML3 and KG-1 cells (both cell lines with low endogenous CD33 expression) to overexpress wild-type CD33 at increasing levels, we found low abundance of CD33 curtailed BMS-986357-induced cytotoxicity. Flow cytometric binding studies identified BMS-986357 to recognize the membrane-distal V-set domain of CD33. Unlike GO, BMS-986357-mediated cytolysis was not affected by over-expression of ABC transporter proteins by AML cells. The potency but not maximal activity of BMS-986357 was reduced by super-physiologic concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact TriNKET ® activity. In the presence of CD33+ AML cells, BMS-986357 activated NK cells but not T cells from healthy donor peripheral blood mononuclear cells and produced similar levels of maximum cytolysis as a CD33/CD3 bispecific antibody. Despite this, BMS-986357 elicited 10 to &amp;gt;100-fold lower soluble cytokine levels including IFN-γ, TNF, IL-6, and GM-CSF than a CD33/CD3 bispecific antibody, suggesting BMS-986357 has the potential for CD33/CD3 bispecific-like efficacy without the associated risk of cytokine release syndrome. Finally, in bone marrow specimens from patients with AML, BMS-986357 killed AML cells but not normal monocytes, suggesting selectivity of drug-induced cytotoxicity toward neoplastic cells, consistent with the notion NK cells are capable to differentiate between healthy and leukemic CD33+ cells. This selectivity suggests BMS-986357 may produce less myelosuppressive toxicity than what has been observed with CD33 targeting antibody-drug conjugates. In summary, our findings demonstrate that BMS-986357 TriNKET ® has potent CD33-dependent cytolytic activity in vitro against human AML cells, supporting the drug's exploration in early phase clinical trials. One such trial (NCT04789655) has opened accrual for adults with relapsed/refractory AML.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

Phenotypic subtypes of leukaemic transformation in chronic myelomonocytic leukaemia.

Chronic myelomonocytic leukaemia (CMML) is a haematological disorder with high risk of transformation to acute myeloid leukaemia (AML). To characterize the phenotypic and genomic patterns of CMML progression, we evaluated a cohort of 189 patients with AML evolving from CMML. We found that transformation occurs through distinct trajectories characterized by genomic profiles and clonal evolution: monocytic (Mo-AML, 53%), immature myeloid (My-AML, 43%) or erythroid (Ery-AML, 2%). Mo-AML, characterized by expansion of monoblasts and promonocytes (low CD34, CD117 expression; high CD14, CD33, CD56 and CD64 expression), were defined by SRSF2, TET2 and RAS pathway mutation co-dominance and were more likely to evolve from SRSF2-TET2 co-mutant CMML through emergence/expansion of RAS pathway mutant clones. Conversely, My-AML, characterized by expansion of immature myeloid blasts (high frequency of CD34, CD38, CD117; low frequencyof CD14, CD64 and CD56 expression) were less likely to exhibit SRSF2-TET2 co-mutations or RAS pathway mutations and had higher frequency of CEBPA mutations. Ery-AML was defined by complex karyotypes and TP53 mutations. Atrend towards improved OS and EFS with hypomethylating agent-venetoclax combination was observed in My-AML, but not Mo-AML. These findings define distinct progression of CMML and set the basis for future studies evaluating the role of phenotype-specific therapeutics.

CD44 is a potential immunotherapeutic target and affects macrophage infiltration leading to poor prognosis

CD44 plays a key role in the communication of CSCs with the microenvironment and the regulation of stem cell properties. UALCAN was used to analyze the expression of CD44 in bladder cancer (BLCA) and normal tissue. The UALCAN was utilized to analyze the prognostic value of CD44 in BLCA. The TIMER database was used to explore the relationship between CD44 and PD-L1; CD44 and tumor-infiltrating immune cells. The regulatory effect of CD44 on PD-L1 was verified by cell experiments in vitro. IHC confirmed the results of the bioinformatics analysis. GeneMania and Metascape were used to analyze protein–protein interaction (PPI) investigations and functional enrichment analysis. We found that BLCA patients with high CD44 expression had worse survival than those with low CD44 expression (P < 0.05). IHC and the TIMER database results showed that CD44 expression was positively correlated with PD-L1 expression (P < 0.05). At the cellular level, the expression of PD-L1 was significantly inhibited after CD44 expression was inhibited by siRNA. Immune infiltration analysis showed that CD44 expression levels in BLCA were significantly correlated with immune infiltration levels of different immune cells. IHC staining results further confirmed that the expression of CD44 in tumor cells was positively associated with the number of CD68+ macrophages and CD163+ macrophages (P < 0.05). Our results suggest that CD44 is a positive regulator of PD-L1 in BLCA and may be a key regulator of tumor macrophages infiltration and may be involved in M2 macrophage polarization. Our study provided new insights into the prognosis and immunotherapy of BLCA patients through macrophage infiltration and immune checkpoints.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-β, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.