Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response.

Introduction: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. Methods: In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. Results and discussion: The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.

Cross-Reactivity to Mutated Viral Immune Targets Can Influence CD8+ T Cell Functionality: An Alternative Viral Adaptation Strategy.

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8+ T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8+ T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8+ T cells were predominantly dual tetramer+, confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8+ T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8+ T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an 'effective' immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNÉ£, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8+ T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.

Single-cell analysis shows that adipose tissue of persons with both HIV and diabetes is enriched for clonal, cytotoxic, and CMV-specific CD4+ T cells.

Persons with HIV are at increased risk for diabetes mellitus compared with individuals without HIV. Adipose tissue is an important regulator of glucose and lipid metabolism, and adipose tissue T cells modulate local inflammatory responses and, by extension, adipocyte function. Persons with HIV and diabetes have a high proportion of CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells in adipose tissue, a subset of which are cytomegalovirus specific, whereas individuals with diabetes but without HIV have predominantly CD69+ CD4+ T cells. Adipose tissue CD69+ and C-G-C+ CD4+ T cell subsets demonstrate higher receptor clonality compared with the same cells in blood, potentially reflecting antigen-driven expansion, but C-G-C+ CD4+ T cells have a more inflammatory and cytotoxic RNA transcriptome. Future studies will explore whether viral antigens have a role in recruitment and proliferation of pro-inflammatory C-G-C+ CD4+ T cells in adipose tissue of persons with HIV.

In chronic infection, HIV gag-specific CD4+ T cell receptor diversity is higher than CD8+ T cell receptor diversity and is associated with less HIV quasispecies diversity.

Cellular immune responses to Gag correlate with improved HIV viral control. The full extent of cellular immune responses comprise both the number of epitopes recognized by CD4+ and CD8+ T cells, as well as the diversity of the T cell receptor (TCR) repertoire directed against each epitope. The optimal diversity of the responsive TCR repertoire is unclear. Therefore, we evaluated the TCR diversity of CD4+ and CD8+ T cells responding to HIV-1 Gag to determine if TCR diversity correlates with clinical or virologic metrics. Previous studies of TCR repertoires have been limited primarily to CD8+ T cell responses directed against a small number of well-characterized T cell epitopes restricted by specific human leucocyte antigens. We stimulated peripheral blood mononuclear cells from 21chronic HIV-infected individuals overnight with a pool of HIV-1 Gag peptides, followed by sorting of activated CD4+ and CD8+ T cells and TCR deep sequencing. We found Gag-reactive CD8+ T cells to be more oligoclonal, with a few dominant TCRs comprising the bulk of the repertoire, compared to the highly diverse TCR repertoires of Gag-reactive CD4+ T cells. HIV viral sequencing of the same donors revealed that high CD4+ T cell TCR diversity was strongly associated with lower HIV Gag genetic diversity. We conclude that the TCR repertoire of Gag-reactive CD4+ T helper cells display substantial diversity without a clearly dominant circulating TCR clonotype, in contrast to a hierarchy of dominant TCR clonotypes in the Gag-reactive CD8+ T cells, and may serve to limit HIV diversity during chronic infection.IMPORTANCE Human T cells recognize portions of viral proteins bound to host molecules (human leucocyte antigens) on the surface of infected cells. T cells recognize these foreign proteins through their T cell receptors (TCRs), which are formed by the assortment of several available V, D and J genes to create millions of combinations of unique TCRs. We measured the diversity of T cells responding to the HIV Gag protein. We found the CD8+ T cell response is primarily made up of a few dominant unique TCRs whereas the CD4+ T cell subset has a much more diverse repertoire of TCRs. We also found there was less change in the virus sequences in subjects with more diverse TCR repertoires. HIV has a high mutation rate, which allows it to evade the immune response. Our findings describe the characteristics of a virus-specific T cell response that may allow it to limit viral evolution.

Group 2 Innate Lymphoid Cells Coordinate Damage Response in the Stomach.

BACKGROUND & AIMS: Severe injury to the lining of the stomach leads to changes in the epithelium (reprogramming) that protect and promote repair of the tissue, including development of spasmolytic polypeptide-expressing metaplasia (SPEM) and tuft and foveolar cell hyperplasia. Acute gastric damage elicits a type-2 inflammatory response that includes production of type-2 cytokines and infiltration by eosinophils and alternatively activated macrophages. Stomachs of mice that lack interleukin 33 (IL33) or interleukin 13 (IL13) did not undergo epithelial reprogramming after drug-induced injury. We investigated the role of group 2 innate lymphoid cells (ILC2s) in gastric epithelial repair. METHODS: Acute gastric injury was induced in C57BL/6J mice (wild-type and RAG1 knockout) by administration of L635. We isolated ILC2s by flow cytometry from stomachs of mice that were and were not given L635 and performed single-cell RNA sequencing. ILC2s were depleted from wild-type and RAG1-knockout mice by administration of anti-CD90.2. We assessed gastric cell lineages, markers of metaplasia, inflammation, and proliferation. Gastric tissue microarrays from patients with gastric adenocarcinoma were analyzed by immunostaining. RESULTS: There was a significant increase in the number of GATA3-positive ILC2s in stomach tissues from wild-type mice after L635-induced damage, but not in stomach tissues from IL33-knockout mice. We characterized a marker signature of gastric mucosal ILC2s and identified a transcription profile of metaplasia-associated ILC2s, which included changes in expression of Il5, Il13, Csf2, Pd1, and Ramp3; these changes were validated by quantitative polymerase chain reaction and immunocytochemistry. Depletion of ILC2s from mice blocked development of metaplasia after L635-induced injury in wild-type and RAG1-knockout mice and prevented foveolar and tuft cell hyperplasia and infiltration or activation of macrophages after injury. Numbers of ILC2s were increased in stomach tissues from patients with SPEM compared with patients with normal corpus mucosa. CONCLUSIONS: In analyses of stomach tissues from mice with gastric tissue damage and patients with SPEM, we found evidence of type 2 inflammation and increased numbers of ILC2s. Our results suggest that ILC2s coordinate the metaplastic response to severe gastric injury.

Changes in Peripheral and Local Tumor Immunity after Neoadjuvant Chemotherapy Reshape Clinical Outcomes in Patients with Breast Cancer.

PURPOSE: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). EXPERIMENTAL DESIGN: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1-high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. RESULTS: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. CONCLUSIONS: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Restricted myeloperoxidase epitopes drive the adaptive immune response in MPO-ANCA vasculitis.

BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.

Adipose Tissue in Persons With HIV Is Enriched for CD4+ T Effector Memory and T Effector Memory RA+ Cells, Which Show Higher CD69 Expression and CD57, CX3CR1, GPR56 Co-expression With Increasing Glucose Intolerance.

Chronic T cell activation and accelerated immune senescence are hallmarks of HIV infection, which may contribute to the increased risk of cardiometabolic diseases in people living with HIV (PLWH). T lymphocytes play a central role in modulating adipose tissue inflammation and, by extension, adipocyte energy storage and release. Here, we assessed the CD4+ and CD8+ T cell profiles in the subcutaneous adipose tissue (SAT) and blood of non-diabetic (n = 9; fasting blood glucose [FBG] < 100 mg/dL), pre-diabetic (n = 8; FBG = 100-125 mg/dL) and diabetic (n = 9; FBG ≥ 126 mg/dL) PLWH, in addition to non- and pre-diabetic, HIV-negative controls (n = 8). SAT was collected by liposuction and T cells were extracted by collagenase digestion. The proportion of naïve (TNai) CD45RO-CCR7+, effector memory (TEM) CD45RO+CCR7-, central memory (TCM) CD45RO+CCR7+, and effector memory revertant RA+(TEMRA) CD45RO-CCR7- CD4+ and CD8+ T cells were measured by flow cytometry. CD4+ and CD8+ TEM and TEMRA were significantly enriched in SAT of PLWH compared to blood. The proportions of SAT CD4+ and CD8+ memory subsets were similar across metabolic status categories in the PLWH, but CD4+ T cell expression of the CD69 early-activation and tissue residence marker, particularly on TEM cells, increased with progressive glucose intolerance. Use of t-distributed Stochastic Neighbor Embedding (t-SNE) identified a separate group of predominantly CD69lo TEM and TEMRA cells co-expressing CD57, CX3CR1, and GPR56, which were significantly greater in diabetics compared to non-diabetics. Expression of the CX3CR1 and GPR56 markers indicate these TEM and TEMRA cells may have anti-viral specificity. Compared to HIV-negative controls, SAT from PLWH had an increased CD8:CD4 ratio, but the distribution of CD4+ and CD8+ memory subsets was similar irrespective of HIV status. Finally, whole adipose tissue from PLWH had significantly higher expression of TLR2, TLR8, and multiple chemokines potentially relevant to immune cell homing compared to HIV-negative controls with similar glucose tolerance.

HLA-A*32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.

BACKGROUND: Vancomycin is a prevalent cause of the severe hypersensitivity syndrome drug reaction with eosinophilia and systemic symptoms (DRESS), which leads to significant morbidity and mortality and commonly occurs in the setting of combination antibiotic therapy, affecting future treatment choices. Variations in HLA class I in particular have been associated with serious T cell-mediated adverse drug reactions, which has led to preventive screening strategies for some drugs. OBJECTIVE: We sought to determine whether variation in the HLA region is associated with vancomycin-induced DRESS. METHODS: Probable vancomycin-induced DRESS cases were matched 1:2 with tolerant control subjects based on sex, race, and age by using BioVU, Vanderbilt's deidentified electronic health record database. Associations between DRESS and carriage of HLA class I and II alleles were assessed by means of conditional logistic regression. An extended sample set from BioVU was used to conduct a time-to-event analysis of those exposed to vancomycin with and without the identified HLA risk allele. RESULTS: Twenty-three subjects met the inclusion criteria for vancomycin-associated DRESS. Nineteen (82.6%) of 23 cases carried HLA-A*32:01 compared with 0 (0%) of 46 of the matched vancomycin-tolerant control subjects (P = 1 × 10-8) and 6.3% of the BioVU population (n = 54,249, P = 2 × 10-16). Time-to-event analysis of DRESS development during vancomycin treatment among the HLA-A*32:01-positive group indicated that 19.2% had DRESS and did so within 4 weeks. CONCLUSIONS: HLA-A*32:01 is strongly associated with vancomycin-induced DRESS in a population of predominantly European ancestry. HLA-A*32:01 testing could improve antibiotic safety, help implicate vancomycin as the causal drug, and preserve future treatment options with coadministered antibiotics.

High CD8 T-Cell Receptor Clonality and Altered CDR3 Properties Are Associated With Elevated Isolevuglandins in Adipose Tissue During Diet-Induced Obesity.

Adipose tissue (AT) CD4+ and CD8+ T cells contribute to obesity-associated insulin resistance. Prior studies identified conserved T-cell receptor (TCR) chain families in obese AT, but the presence and clonal expansion of specific TCR sequences in obesity has not been assessed. We characterized AT and liver CD8+ and CD4+ TCR repertoires of mice fed a low-fat diet (LFD) and high-fat diet (HFD) using deep sequencing of the TCRß chain to quantify clonal expansion, gene usage, and CDR3 sequence. In AT CD8+ T cells, HFD reduced TCR diversity, increased the prevalence of public TCR clonotypes, and selected for TCR CDR3 regions enriched in positively charged and less polarized amino acids. Although TCR repertoire alone could distinguish between LFD- and HFD-fed mice, these properties of the CDR3 region of AT CD8+ T cells from HFD-fed mice led us to examine the role of negatively charged and nonpolar isolevuglandin (isoLG) adduct-containing antigen-presenting cells within AT. IsoLG-adducted protein species were significantly higher in AT macrophages of HFD-fed mice; isoLGs were elevated in M2-polarized macrophages, promoting CD8+ T-cell activation. Our findings demonstrate that clonal TCR expansion that favors positively charged CDR3s accompanies HFD-induced obesity, which may be an antigen-driven response to isoLG accumulation in macrophages.

Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

Select CMV epitopes drive life-long CD8+ T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4+ T cells specific for human CMV (HCMV) are elevated in HIV+ HCMV+ subjects. To determine whether HCMV epitope-specific CD4+ T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4+ T cells in coinfected HLA-DR7+ long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4+ T cells were inflated among these HIV+ subjects compared with those from an HIV- HCMV+ HLA-DR7+ cohort or with HLA-DR7-restricted CD4+ T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4+ T cells consisted of effector memory or effector memory-RA+ subsets with restricted TCRß usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX3CR1, CD38, or HLA-DR but less often coexpressed CD38+ and HLA-DR+ The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4+ T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease.

Greater activation of peripheral T follicular helper cells following high dose influenza vaccine in older adults forecasts seroconversion.

BACKGROUND: Influenza related morbidity and mortality disproportionately impacts older adults. The serologic response to vaccine is diminished in older adults; however, high dose inactivated influenza vaccine (HD IIV) has shown improved rates of seroconversion compared to standard dose (SD IIV). We hypothesize this may be due to the superior ability of high dose vaccine to activate T follicular helper (Tfh) cells and provide B cell dependent T cell help. METHODS: We measured peripheral Tfh (pTfh) activation in 50 community dwelling adults 65years or older who were randomly assigned to receive either the HD IIV or SD IIV. RESULTS: The HD vaccination elicited significantly higher levels of ICOS expression on pTfh cells, at day 7 compared to SD vaccination (p=0.02). The magnitude of the increase in ICOS+ pTfh cells from baseline to day 7 was predictive of seroconversion for both influenza A and B vaccination. CONCLUSION: Strong Tfh activation in response to influenza vaccination forecasts successful seroconversion in older adults, and HD IIV elicits greater Tfh activation than SD IIV. Future vaccine studies should focus on ways to further optimize the Tfh response.

Fulminant Myocarditis with Combination Immune Checkpoint Blockade.

Immune checkpoint inhibitors have improved clinical outcomes associated with numerous cancers, but high-grade, immune-related adverse events can occur, particularly with combination immunotherapy. We report the cases of two patients with melanoma in whom fatal myocarditis developed after treatment with ipilimumab and nivolumab. In both patients, there was development of myositis with rhabdomyolysis, early progressive and refractory cardiac electrical instability, and myocarditis with a robust presence of T-cell and macrophage infiltrates. Selective clonal T-cell populations infiltrating the myocardium were identical to those present in tumors and skeletal muscle. Pharmacovigilance studies show that myocarditis occurred in 0.27% of patients treated with a combination of ipilimumab and nivolumab, which suggests that our patients were having a rare, potentially fatal, T-cell-driven drug reaction. (Funded by Vanderbilt-Ingram Cancer Center Ambassadors and others.).

CD70 Exacerbates Blood Pressure Elevation and Renal Damage in Response to Repeated Hypertensive Stimuli.

RATIONALE: Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of memory T cells, which requires the costimulatory molecule CD70 on antigen-presenting cells, is a cardinal feature of adaptive immunity. OBJECTIVE: To test the hypothesis that CD70 and immunologic memory contribute to the blood pressure elevation and renal dysfunction mediated by repeated hypertensive challenges. METHODS AND RESULTS: We imposed repeated hypertensive challenges using either N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)/high salt or repeated angiotensin II stimulation in mice. During these challenges effector memory T cells (T(EM)) accumulated in the kidney and bone marrow. In the L-NAME/high-salt model, memory T cells of the kidney were predominant sources of interferon-γ and interleukin-17A, known to contribute to hypertension. L-NAME/high salt increased macrophage and dendritic cell surface expression of CD70 by 3- to 5-fold. Mice lacking CD70 did not accumulate T(EM) cells and did not develop hypertension to either high salt or the second angiotensin II challenge and were protected against renal damage. Bone marrow-residing T(EM) cells proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred T(EM) cells from hypertensive mice homed to the bone marrow and spleen and expanded on salt feeding of the recipient mice. CONCLUSIONS: Our findings illustrate a previously undefined role of CD70 and long-lived T(EM) cells in the development of blood pressure elevation and end-organ damage that occur on delayed exposure to mild hypertensive stimuli. Interventions to prevent repeated hypertensive surges could attenuate formation of hypertension-specific T(EM) cells.

RAS/MAPK Activation Is Associated with Reduced Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer: Therapeutic Cooperation Between MEK and PD-1/PD-L1 Immune Checkpoint Inhibitors.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. EXPERIMENTAL DESIGN: We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1-targeted immunotherapy in mouse models of breast cancer. RESULTS: Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras-MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer. CONCLUSIONS: These data suggest the possibility that Ras-MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1-targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors.