Generation of an optimal T cell therapeutic expressing high frequencies of transgenic T cell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRab reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRab was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating T cell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 T cells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited T cell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRab chains can be incorporated in current T cell production protocols and is preferential to ensure an improved and safe clinical therapeutic.

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Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP–restricted CD4 + T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65 (142–158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

Detection of SARS-coronavirus-2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relative high frequency and prevalence of cross-reactive T cells, we hypothesized CMV may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved PBMCs with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.

SARS-CoV-2 vaccine immunogenicity is commonly evaluated by measuring antibody titers against the SARS-CoV-2 Spike (S) protein. Previously, inferior humoral vaccination responses in patients with lymphoid malignancies have been shown. 1 This can be attributed to immune defects caused by disease or treatment. NHL and CLL treated with CD20-and MM with CD38-directed therapies, leads to longlasting B-or plasma-cell depletion, respectively. CLL and MM are associated with hypogammaglobulinemia and aberrations in T-cell function. T-cell immunity is vital for viral clearance and long-lasting protection against COVID-19 after vaccination. 2 Moreover, high CD8 + T-cells contribute to COVID-19 survival in hematological patients. 3 Studies investigating T-cell responses after vaccination in patients with lymphoid malignancies are, however, scarce and results are conflicting. This leaves a knowledge gap, underlining the urgency of an in-depth and reproducible analysis of functional SARS-CoV-2 specific T-cell responses following vaccination in hematological patients. Patients. Adult patients diagnosed with CLL, NHL, or MM at two tertiary care centers in the Netherlands undergoing SARS-CoV-2 vaccination were included in the study. Serological and T-cell responses

Virus-specific cellular and humoral responses are major determinants for protection from critical illness after SARS-CoV-2 infection. However, the magnitude of the contribution of each of the components to viral clearance remains unclear. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 102 hospitalised patients with moderate and severe COVID-19 in a longitudinal design. Delayed viral clearance was associated with more severe disease and was associated with higher levels of SARS-CoV-2-specific (neutralising) antibodies over time, increased numbers of neutrophils, monocytes, basophils, and a range of pro-inflammatory cyto-/chemokines illustrating ongoing, partially Th2 dominating, immune activation. In contrast, early viral clearance and less critical illness correlated with the peak of neutralising antibodies, higher levels of CD4 T cells, and in particular naïve CD4+ T cells, suggesting their role in early control of SARS-CoV-2 possibly by proving appropriate B cell help. Higher counts of naïve CD4+ T cells also correlated with lower levels of MIF, IL-9, and TNF-beta, suggesting an indirect role in averting prolonged virus-induced tissue damage. Collectively, our data show that naïve CD4+ T cell play a critical role in rapid viral T cell control, obviating aberrant antibody and cytokine profiles and disease deterioration. These data may help in guiding risk stratification for severe COVID-19.

It is not exactly clear yet which type of immune response prevails to accomplish viral clearance in coronavirus disease 2019 (COVID-19). Studying a patient with chronic lymphocytic leukemia and hypogammaglobulinemia who suffered from COVID-19 provided insight in the immunological responses after treatment with COVID-19 convalescent plasma (CCP). Treatment consisted of oxygen, repeated glucocorticosteroids and multiple dosages of CCP guided by antibody levels. Retrospectively performed humoral and cellular immunity analysis made clear that not every serological test for COVID-19 is appropriate for follow-up of sufficient neutralizing antibodies after CCP. In retrospect, we think that CCP merely bought time for this patient to develop an adequate cell immune response which led to viral clearance and ultimately clinical recovery.

SARS-CoV-2 viral load is associated with disease severity. A better understanding of immunological mechanisms involved in viral clearance is crucial to guide new therapeutic strategies. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 150 hospitalized COVID-19 patients. Delayed viral clearance was associated with more severe disease, which occurred after the virus had been cleared in most cases. Paradoxically, delayed viral clearance was associated with over time higher maximum levels of SARS-CoV-2 specific IgG, IgA, and neutralizing antibodies, increased numbers of eosinophils, monocytes, and pro-inflammatory cyto-/chemokines. In contrast, early viral clearance and less critical illness correlated with higher levels of CD4 + and CD8 + T cells. Collectively, our data show that absence of rapid T cell control corresponds with delayed clearance and aberrant antibody and cytokine profiles. Viral clearance often precedes critical illness, which suggests immunopathology as underlying mechanism. These data can guide treatment strategies.