While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.

IL-21 is produced predominantly by activated CD4+ T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γc) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8+ T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4+ and CD8+ T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R−/− mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R−/− T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.

CD8 + T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8 + T cell responses, therapeutic approaches to generate Ag-specific CD8 + T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8 + T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ~1000-fold more potent at eliciting CD8 + T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8 + T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8 + T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8 + T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8 + T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

BackgroundAntigen-specific CD8+ T cell activity is critical for mounting an effective immune response in a wide range of indications, including immune-oncology and infectious diseases.MethodsTo elicit antigen-specific CD8+ T cell activity, we used microfluidics cell squeezing (Cell Squeeze®) to deliver antigens directly to the cytosol of antigen presenting cells (APCs). Direct cytosolic delivery bypasses the need for cross-presentation and efficiently loads antigen into the major histocompatibility complex class I (MHC-I) pathway. The Cell Squeeze® platform is generally agnostic to cell type and material. Therefore, not only does microfluidic squeezing enable cell subsets within human peripheral blood mononuclear cells (PBMCs) to function as unconventional APCs, but it also enables us to efficiently investigate a wide range of antigens including whole protein, peptides, and mRNA. This ‘plug and play’ nature of the platform allows for broad application in multiple disease areas.ResultsIn human cells, we demonstrated that microfluidic squeezing of PBMCs enables effective delivery to the major cell subsets including T cells, B cells, NK cells and monocytes. Delivery of CMV and HPV16 synthetic long peptides (SLPs) resulted in robust in vitro responses of both CD8+ T cell clones and patient-derived memory populations. To broaden the impact of our PBMC-based cell therapy approach, we investigated several other antigens relevant to other disease areas. Additional materials we delivered via squeezing and demonstrated antigen presentation include neoantigens, M1 Influenza mRNA, and pp65 SLP. Cell Squeeze® platform is simple to use and amenable to scale up. We demonstrated that delivery and viability for research scale process (~2 × 106 cells) is equivalent to delivery and viability of PBMCs processed at manufacturing scale (~1 × 109 cells).ConclusionsMicrofluidic cell squeezing of human PBMCs with antigenic material can be tailored to produce APCs that drive robust CD8+ T cell response against targets across multiple disease areas and has been scaled up for clinical use. SQZ-PBMC-HPV are currently under clinical evaluation for treatment of HPV16+ tumors.

Tuberculosis is a pulmonary disease, caused by Mycobacterium tuberculosis (Mtb) infection, which affects approximately one third of the global population and is a major public health concern. Although T cells are essential in mediating protection against Mtb infection, they are often unable to sterilize infected tissues. One possible reason for an ineffective immune response is the onset of T cell exhaustion or dysfunction. T cell exhaustion is characterized by the progressive loss of T cell effector function accompanied by an increase in expression of inhibitory receptors. Here we show that during Mtb infection, T cells express the inhibitory molecules Tim3 and PD1, and T cells that express both Tim3 and PD1 have decreased IFNγ and TNF production. In addition, antibody blockade of Tim3 improves T cell function and decreases bacterial burden. These results indicate that T cells are functionally exhausted during chronic Mtb infection and that blockade of inhibitory receptors can re-invigorate T cell function and improve the outcome of infection. Based on a possible role for CEACAM1 in the T cell inhibitory signal mediated by Tim3, we are currently performing experiments to determine how CEACAM1 affects host defense against Mtb. Understanding the roles of inhibitory receptors, such as Tim3 and PD1, during Mtb infection is providing insight into why T cell immunity is insufficient to eradicate Mtb infection.

BackgroundCharacterization of human immune responses by profiling immune cells from patients is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, identify pharmacodynamic/response biomarkers, and guide patient selection strategies. Extensive immune cell heterogeneity necessitates comprehensive high parameter immunophenotyping to yield these actionable insights.MethodsCytometry by time-of-flight (CyTOF) was performed on homogenates from commercially procured tumors (n=28) and matched PBMCs (n=7) from patients with various solid tumors (colon (n=10), endometrial (n=9), kidney (n=4), liver (n=2), skin (n=1), lung (n=1), and gastro-intestinal (n=1)). Two antibody panels, recognizing a total of 18 lineage and 31 target proteins, were used to profile marker expression among the major lymphocyte and myeloid lineages. Data were analyzed using manual gating and non-linear dimensionality reduction (tSNE and UMAP), and expression was measured by frequency (% gate) and arcsinh-transformed median ion counts. Pairwise Wilcoxon Rank Sum tests were performed on arcsinh-transformed median ion counts to determine statistically significant differences in marker expression, and P values were adjusted using Benjamini-Hochberg correction (p<0.05 considered statistically significant). Cell subpopulation percentages were compared using unpaired two-sided T-tests. Sample populations with less than 150 events were excluded from analysis. The initial analysis focused on CD8 T cells as a primary mediator of antitumor immunity.ResultsMatched samples revealed enrichment of effector memory (EM) and central memory (CM) CD8 T cells in tumors compared to PBMCs, as expected. EM cells represented on average 63.36% of the CD8 T cells in tumors vs 30.31% in PBMCs (p=0.0067), and CM cells 12.11% vs 5.58% respectively (p=0.1558). Non-linear dimensionality reduction mapping of these CD8 EM and CM cell subtypes among tumors displayed an activated but potentially dysfunctional phenotype, characterized by substantially higher expression of multiple coinhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT) and activation markers (HLA-DR, ICOS) compared to PBMCs. Among these cells, a PD-1+/LAG-3+ subset, observed in 17/28 TIL samples, expressed TIM-3, TIGIT, HLA-DR, and ICOS at significantly higher levels compared to other PD1/LAG3 expression subsets. Interestingly, CD137 (4-1BB), a marker of potentially tumor-reactive cells, is expressed predominantly in PD-1+ memory CD8 T cells, with the most intense expression levels observed in the PD-1+/LAG-3+ subset.ConclusionsThe present results provide insight into the relative (co)expression of potentially targetable immunological pathways, and suggest a biological basis for informing approaches to combination checkpoint inhibition therapy.AcknowledgementsWe thank Paul Fischer for his contributions in acquiring the CyTOF data and performing initial data QC and analysis.Ethics ApprovalThis study was approved by Bristol Myers Squibb’s Global Data Repository (Biological Assessment of Risk (BAR) number LVL_2020_12339). Samples were provided by Discovery Life Sciences (CA), MT Group (CA), Avaden BioSciences (WA), or BioOptions (CA). All patients gave written informed consent at the time of sample collection according to the IRB protocols of each provider.

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