Given the scarcity of effective therapeutic targets, metastatic triple negative breast cancer (mTNBC) has shorter survival times compared to other advanced breast cancer subtypes. Although chemo-immunotherapy with immune checkpoint inhibitors (ICIs) in PD-L1+ mTNBC has shown promise, survival benefit remains modest. Therefore, it is crucial to gain improved insight into the mechanisms underlying response and resistance to checkpoint inhibition in mTNBC.
We employed single cell RNA sequencing (scRNAseq), single cell secretomics, and flow cytometry to identify transcriptomic and proteomic peripheral immune cell signatures associated with response and non-response to anti-PD-1/PD-L1 therapy and chemotherapy in mTNBC.
Transcriptomic analysis reveal divergent transcriptional programming of CD33+ myeloid cells between responders and non-responders, even in pretreatment PBMC samples. This divergence, in responders, is characterized by an immune-promoting CD33+ cell phenotype involving IL1b signaling compared to non-responders, where an immunosuppressive phenotype marked by IL1b inhibition is observed. These baseline differences become more pronounced during the course of chemo-immunotherapy. Differences in CD33+ cell phenotype result in functional differences in lymphocyte activities between responders and non-responders. Depletion of CD33+ cells in pre-treatment samples from non-responders, restores T cell effector function.
Our findings highlight CD33+ cell phenotype as a key determinant of response to chemo-immunotherapy, which can be assessed from peripheral blood. This offers a valuable tool in the context of metastatic TNBC, in which tissue sampling is often challenging.
© 2025. The Author(s).

Antibody conjugates are the foundation of a wide range of diagnostic and therapeutic applications. Although many antibody-conjugation techniques are robust and efficient, obtaining homogeneous multimeric conjugation products remains challenging. Here we report a modular and versatile technique for the site-directed multivalent conjugation of antibodies via the small-protein ubiquitin. Specifically, multiple ubiquitin fusions with antibodies, antibody fragments, nanobodies, peptides or small molecules such as fluorescent dyes can be conjugated to antibodies and nanobodies within 30 min. The technique, which we named 'ubi-tagging', allowed us to efficiently generate a bispecific T-cell engager as well as nanobodies conjugated to dendritic-cell-targeted antigens that led to potent T-cell responses. Using both recombinant ubi-tagged proteins and synthetic ubiquitin derivatives allows for the iterative, site-directed and multivalent conjugation of antibodies and nanobodies to a plethora of molecular moieties.
© 2025. The Author(s).

Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck.
The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging, and flow cytometry. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA.
Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and non-transduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Interestingly, oxamic acid was found to inhibit CD64-CR T cell proliferation, indicating the involvement of lactate. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger antibody-dependent cellular cytotoxicity (ADCC) in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression in cancer cells via IFNγ. PD-L1 positive SCCHN cells in combination with anti-PD-L1 mAb and CD64-CR T cells were killed by ADCC, which enhanced direct cytotoxicity. These findings indicate that the glycolytic phenotype is involved in CD64-CR T cell proliferation/expansion. These cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells.
CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and SCCHN and the identification of novel FcγR ALs in cancer and healthy cells.
© 2025. The Author(s).

HIV persists in diverse tissues, with distinct cellular reservoirs presenting a major barrier to a cure and requiring targeted therapeutic strategies to address this heterogeneity. Here, we developed tissue models of HIV latency using human tonsillar, intestinal and cervicovaginal tissues. These models revealed differential HIV infection across CD4+ T cell subpopulations, with ART partially restoring CD4+ T cells and reducing intact HIV DNA. T follicular helper cells (T FH CD69+ CCR7- ) were the primary inducible reservoir in tonsils, while tissue-resident memory cells (T RM CD69+ CD49a+ ) dominated in the intestine. Identification of markers for inducible reservoirs revealed that CD69, CD45RO, and PD-1 were shared across tissues, while CXCR5 in the tonsils and CD49a in the intestine served as tissue-specific markers. Furthermore, using different latency reversal agents (LRAs) we found that Histone Deacetylase Inhibitors (HDACis) failed to induce HIV in any tissue, the SMAC mimetic AZD5582 was effective only in a resident-memory CD4+ T cell subpopulation in the intestine, and IL15 exhibited the broadest reactivation potential across tissues and CD4+ T subsets. These models recapitulate key aspects of HIV infection providing insights into the inducible reservoir's composition in different tissues and informing strategies for its elimination.

The current study explores the impact of CLL on γδ T cells and, in an attempt to better understand the sources of immunosuppression, assesses the impact of M-MDSCs on γδ T cells in vitro.
The study included 163 CLL patients and 34 healthy volunteers. γδ T cells were screened with flow cytometry, including NKG2D, Fas, FasL, and TRAIL staining. Additionally, to deepen understanding of the immunosuppressive impact of CLL on γδ T, a set of in vitro co-cultures of γδ T and M-MDSCs was performed.
RNAseq revealed significant, though relatively minor, changes in the transcriptome. Functional analyses showed a minor drop in cytotoxic potential against CLL cells. Finally, depletion of M-MDSCs from CLL-derived peripheral blood mononuclear cells did not restore γδ T cells' proliferative response.
Altogether, this suggests a minor impact of M-MDSCs on activated γδ T. Thus, it seems probable that other mechanisms than M-MDSCs mediate the negative impact of CLL on circulating γδ T cells.