Low Numbers Of CD4 Research Articles

Fusobacterium nucleatum, immune responses, and metastatic organ diversity in colorectal cancer liver metastasis.

The presence of Fusobacterium nucleatum is associated with an immunosuppressive tumor immune microenvironment (TIM) in primary colorectal cancer (CRC), contributing to tumor progression. Its persistence in CRC liver metastasis tissues raises questions about its role in modulating local and systemic immune responses and influencing recurrence patterns. This retrospective cohort study of 218 patients with CRC liver metastasis investigated the association of F. nucleatum in CRC liver metastasis tissues with systemic inflammation, TIM alterations, and the number of metastatic organs involved in recurrence. Two-step polymerase chain reaction (PCR), including digital PCR, detected F. nucleatum in 42% (92/218) of fresh-frozen specimens of CRC liver metastases. Compared with the F. nucleatum-none group, the F. nucleatum-high group showed higher C-reactive protein levels (0.82 vs. 0.22 mg/dL; Ptrend = 0.02), lower numbers of CD8+ cells (33.2 vs. 65.3 cells/mm2; Ptrend = 0.04) and FOXP3+ cells (11.3 vs. 21.7 cells/mm2; Ptrend = 0.01) in the TIM, and a greater number of metastatic organs involved in recurrence (1.6 vs. 1.1; p < 0.001). The presence of F. nucleatum in CRC liver metastasis tissues was associated with increased systemic inflammation, TIM alterations, and a greater number of metastatic organs involved in recurrence. These findings suggest a potential contribution of F. nucleatum to the metastatic propensity of CRC cells and could inform future research to enhance understanding of the interaction between tumor, host, and microbes in the metastatic process.

Immunodeviation towards T cell-mediated immune response in the testes of LPS-induced mouse epididymo-orchitis

The testicular consequences of acute epididymo-orchitis remain largely unelucidated in long-term damage, which might be a neglected factor for male infertility. In this study, the differential phenotype of testicular immune cell subpopulations in lipopolysaccharide (LPS)-induced mouse epididymo-orchitis were analyzed by flow cytometry on day 1, day 7, and day 28. The number of macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) steadily decreased in the testes with inoculation. Total F4/80-CD11c+ dendritic cells (DCs) maintained a relatively stable level, whereas conventional type 1 dendritic cells (cDC1) increased gradually from day 1 to day 28. There was a lower number of CD4+ and CD8+ T cells at day 1 and day 7, and they had similar results with a ceiling level at day 28. The testes displayed a higher level of CD3+ T cells but a lower frequency of macrophages, cDC2, and neutrophils at 28 days post-inoculation compared with the epididymis. In summary, our data indicates acute epididymo-orchitis could lead to long-term damage in the testes, which is characterized by CD3+ T cell (including CD4+ and CD8+ T cells)-mediated immune responses.

Efficacy of lymphatic pump treatment in conjunction with methotrexate on rats with adjuvant-induced arthritis

Abstract Rheumatoid Arthritis (RA) is an autoimmune disease targeting synovial joints in which self-reactive T cells release cytokines and B cells produce self-reactive antibodies resulting in an inflammatory response. Lymphatic pump treatment (LPT) promotes lymphatic flow resulting in a flushing out of the immune-complexes and cytokines. A rat mode on RA (AIA) was used to determine whether LPT in conjunction with the anti-rheumatic drug, methotrexate (MTX), enhanced its therapeutic impact on RA. A preventative study was performed in which MTX was given along with LPT (3 times a day for 7 days) . Ankles were assessed daily for signs of inflammation. Flow cytometry was performed on spleen and popliteal lymph nodes (PLN) to determine changes in leukocyte populations. Cytokine levels were determined on ankle lysates. The study showed that MTX treatment delayed the onset of arthritis. MTX reduced the spleen weights (p&amp;lt;0.05). LPT also reduced spleen weights but did not enhance the MTX reduction. LPT reduced PLN weights compared to MTX. MTX resulted in higher numbers of CD4+ T cells and lower numbers of CD8+ T cells and B cells in spleens. PLNs treated with MTX had reduced lymphocyte numbers. MTX treatment reduced CINC-1 and TNFa levels (p&amp;lt;0.05). LPT also reduced CINC-1 levels. In conclusion, MTX treatments delayed arthritis formation in AIA rats while LPT affected both spleen and PLN weights. Thus, the addition of LPT to RA treatment showed potential benefits without negative effects.

Abstract 5528: Adaptive immune cells in colorectal cancer between normal mucosa, primary tumor and liver metastasis

Abstract Introduction: We aimed to assess the role of local adaptive immunity in the metastatic process by comparing densities of adaptive immune cells between normal colorectal mucosa (NM), primary colorectal cancer (pCRC) and liver metastases (LM) in patients with synchronous and metachronous disease. Patients and Methods: We enrolled patients, who underwent resection of pCRC and LM in Pilsen University Hospital between 1999 and 2022. 56 patients presented with LM (stage IV, synchronous) and 43 patients (stage II/III) developed LM later (metachronous). After immunohistochemical staining and whole slide scanning densities of CD3+ and CD8+ T cells and CD20+ B cells were quantified in NM, pCRC and LM using QuPath software. In pCRC and LM cell densities were measured in tumor center (TC), inner margin (IM), outer margin (OM) and peritumor (PT) region. IM and OM were defined as 500 µm on each side of the invasive tumor border. Results: For all examined cells in both groups we found smaller densities in TC of pCRC and LM compared to NM (Table). In pCRC of synchronous group compared to LM densities of all cells were smaller in IM and OM and densities of CD3+ and CD8+ cells were smaller in PT region. In pCRC of metachronous group compared to LM densities of all cells were greater in TC, densities of CD3+ were smaller in OM and PT region, whereas densities of CD20+ cells were smaller only in OM. Compared to metachronous group patients with synchronous metastases had smaller densities of CD3+ T cells in IM and CD8+ T cells in TC and IM of pCRC. Conclusion: Development of pCRC is accompanied by decreased densities of T and B cells in TC compared to NM, which further decrease in metachronous LM. Lower densities of T and B cells in IM and OM and T cells in PT region of pCRC compared to LM is a hallmark of synchronous group. TC of pCRC harbors larger numbers of T and B cells compared to LM in metachronous group. Low numbers of CD3+ and CD8+ T cells in IM and CD8+ T cells in TC of primary CRC may contribute to development of synchronous metastases. Densities of CD3+, CD8+ and CD20+ cells per mm2 of the tissue (median) Patients witn liver metastases Synchronous Metachronous NM pCRC LM NM pCRC LM CD3 NM 1014 1042 CD3 TC 313p&amp;lt;0.05 to NM 249p&amp;lt;0.05 to NM 338p&amp;lt;0.05 to NM 199p&amp;lt;0.05 to NM and pCRC CD3 IM 252 431p&amp;lt;0.05 to pCRC 393p&amp;lt;0.05 to pCRC in synchronous group 483 CD3 OM 521 1437p&amp;lt;0.05 to pCRC 649 1953p&amp;lt;0.05 to pCRC CD3 PT 479 873p&amp;lt;0.05 to pCRC 447 987p&amp;lt;0.05 to pCRC CD8 NM 319 390 CD8 TC 58p&amp;lt;0.05 to NM 52p&amp;lt;0.05 to NM 102p&amp;lt;0.05 to NM and to pCRC in synchronous group 52p&amp;lt;0.05 to NM and pCRC CD8 IM 65 96p&amp;lt;0.05 to pCRC 102p&amp;lt;0.05 to pCRC in synchronous group 120 CD8 OM 206 559p&amp;lt;0.05 to pCRC 310 363 CD8 PT 177 369p&amp;lt;0.05 to pCRC 218 291 CD20 NM 331 215 CD20 TC 32p&amp;lt;0.05 to NM 11p&amp;lt;0.05 to NM 27p&amp;lt;0.05 to NM 12p&amp;lt;0.05 to NM and pCRC CD20 IM 20 39p&amp;lt;0.05 to pCRC 20 33 CD20 OM 137 327p&amp;lt;0.05 to pCRC 134 303p&amp;lt;0.05 to pCRC CD20 PT 153 224 196 213 Citation Format: Andriy Trailin, Wenjing Ye, Esraa Ali, Sergii Pavlov, Lenka Cervenkova, Filip Ambrozkiewicz, Ondrej Vycital, Ondrej Daum, Vaclav Liska, Kari Hemminki. Adaptive immune cells in colorectal cancer between normal mucosa, primary tumor and liver metastasis [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 5528.

Abstract IA007: Distinct immune landscapes characterize highly and minimally invasive brain metastases

Abstract IA007: Distinct immune landscapes characterize highly and minimally invasive brain metastases

Abstract B023: The role of ribosomal protein L22 in regulating the immune landscape in mismatch repair deficient endometrial cancers

Abstract The purpose of this study is to identify novel mechanisms of immune evasion in mismatch repair deficient endometrial cancers. Despite having a hypermutated phenotype that is proposed to lead to the accumulation of many neoantigens for immune recognition, our group has found that mismatch repair deficient endometrial cancers characterized by MLH1 protein loss display large variations in the number of tumors infiltrating CD8+ T cells (TILs) across different tumors. To better understand how variations in TILs arise in mismatch repair deficient endometrial cancers, we utilized human endometrial cancer samples from the UNCseq cohort. Immunohistochemistry (IHC) was used to identify expression patterns in proteins of interest across mismatch repair deficient endometrial cancers. To determine how expression patterns of proteins of interest correlate with TILs in our patient samples, Definiens Tissue Studio was utilized to quantify immunofluorescent CD8+ TILs in our mismatch repair deficient endometrial cancer cohort. Although upregulation of programmed death ligand 1 (PD-L1) expression is often cited as an explanation for low TILs, analysis of human data by our group shows that PD-L1 expression does not correlate with TIL numbers in mismatch repair deficient endometrial cancers. Interestingly, The Cancer Genome Atlas (TCGA) data reported that ribosomal protein L22 (RPL22) is mutated in ~50% of mismatch repair deficient endometrial cancers. We analyzed RPL22 by IHC and found that it was lost in 52% of our human mismatch repair deficient endometrial cancer cohort. Loss of various ribosomal proteins can alter translation efficiency of proteins critical for promoting immunosurveillance and the immune response in cancer. In our cohort, mismatch repair deficient endometrial cancers that have lost expression of RPL22 have lower numbers of CD8+ TILs. Preliminary data also show that human endometrial cancer cell lines that do not express RPL22 also do not express beta-2-microglobulin (β2M), a component of the major histocompatibility complex I (MHC-I) molecules that is necessary for neoantigen presentation to CD8+ T cells. Thus, we hypothesize that RPL22 loss in mismatch repair deficient endometrial cancers results in decreased translation of β2M and CD8+ T cell evasion. Moving forward, we speculate that RPL22 could be utilized as a predictive biomarker for immunotherapy response in mismatch repair deficient endometrial cancers. Citation Format: Macy L. Osborne, Hannah Atkins, Andrew B. Gladden, Russell R. Broaddus. The role of ribosomal protein L22 in regulating the immune landscape in mismatch repair deficient endometrial cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B023.

Lymphocyte subsets and soluble forms of MIC-A and MIC-B are prognostic factors in non-Hodgkin lymphoma patients.

MIC-A and MIC-B are the natural ligands for NKG2D, an activator receptor expressed in NK cells. Soluble isoforms of MIC-A and MIC-B (sMICA, sMICB) have been identified in different malignancies, affecting NK cells' cytotoxicity. The study was performed to determine the levels of sMICA, sMICB, the expression of MIC-A, and MIC-B on tumor tissues, and lymphocyte subpopulations (CD4 + , CD8 + , NK, NKT, Tγδ cells, B cells, monocytes) in 94 patients with non-Hodgkin's lymphoma (NHL) and 72 healthy donors.The most frequent lymphoma was diffuse large B cell lymphoma (48%). Patients with NHL had decreased numbers of CD4 T cells, CD8 T cells, B cells, monocytes, NK cells, type 1 dendritic cells, γδ T cells, and increased iNKT cells. Patients showed higher levels of sMIC-A and similar serum levels of sMIC-B.Survival was poorer in patients having higher LDH values and lower numbers of CD4 T cells, type 1 dendritic cells, gamma-delta T cells, and high levels of sMIC-A.In conclusion, high levels of sMIC and decreased numbers in circulating lymphocyte subsets are related to poor outcomes in NHL.

Itacitinib for Prevention of Graft-Versus-Host Disease and Cytokine Release Syndrome with T-Cell Replete Peripheral Blood Haploidentical Transplantation

Introduction: Haploidentical peripheral blood allogeneic hematopoietic cell transplantation (PB haplo-HCT) can be complicated by graft-versus-host disease (GVHD) and cytokine release syndrome (CRS). Acute GVHD rates are higher with PB grafts compared with bone marrow, affecting 35-45% of patients and outcomes are poor in steroid refractory cases. Severe CRS occurs in 10-15% of patients receiving PB haplo-HCT and is associated with high non-relapse mortality and one-year overall survival between 25-30%. As interferon-γ and IL-6 are important mediators in both acute GVHD and CRS, we hypothesized that JAK1 inhibition with itacitinib could prevent these toxicities without impairing engraftment. Here we report outcomes from our study of itacitinib with haplo-HCT (NCT03755414). Methods: Patients with AML, MDS, ALL, or NHL in remission undergoing PB haplo-HCT were treated with itacitinib 200 mg/day on days -3 through +100 (first 20 patients) or 180 (second 22 patients), followed by a taper. Myeloablative and reduced intensity conditioning were allowed. GVHD prophylaxis was tacrolimus, mycophenolate mofetil, and post-transplant cyclophosphamide. Primary outcomes were incidence of primary graft failure and incidence of grade III-IV acute GVHD. Secondary outcomes included incidence and severity of CRS, relapse, GRFS, and overall survival. Results: Forty-two patients were enrolled and underwent transplantation on study between November 2019 and December 2022, median days of follow up was 453 days (range 142-1259). The patient population had a median age of 60 years (20 - 73 years) with the majority of patients having AML (AML 60%, ALL 21%), MDS 10%, MF 5%, NHL 4%). All patients with AML, MDS, or ALL were in a complete morphologic remission, although 45% had disease by either cytogenetics, next generation sequencing, or measurable residual disease assay (flow cytometry for AML/MDS, NGS for ALL). There were no cases of grade III-IV acute GVHD. The cumulative incidence of grade II acute GVHD on day 100 was 17.3% (95% CI, 7% - 31%), day 180 was 20.5% and day 365 was 20.5%. There were no cases of severe CRS with 90% of patients having grade 1 CRS and 10% having no CRS. No anti-IL6R or steroid therapy was used. There were no cases of primary graft failure. The cumulative incidence of relapse at 1 year was 10% (95% CI, 3% to 22%) and at 2 years was 14% (95% CI, 5% to 29%). In patients who survived without relapse to day 100, the 1-year cumulative incidence of moderate or severe chronic GVHD was 5% (95% CI 1% - 17%). The refined GRFS at day +180 was 85% (95% CI 69% - 93%) and at day +365 was 79% (95% CI 62% - 89%). Overall survival at 180 days 87% (95% CI 72% - 94%) and at 1 year was 80% (95% CI 63% - 90%). Analysis of peripheral blood mononuclear cells at day +28, +60, and +100, revealed that patients on itacitinib, compared with control haplo-HCT patients, had lower numbers of CD4 and CD8 central memory T cells, lower numbers of CD4 and CD8 naïve T cells, and higher numbers of classical monocytes, nonclassical monocytes, and both myeloid and plasmacytoid dendritic cells. In itacitinib treated patients, T-cell subsets expressed lower LAG3 and PD-1 and higher TIGIT, while monocytes expressed higher HLA-DR, CD80, and CD86. Conclusions: Itacitinib with PB haplo-HCT was safe with low rates of acute and chronic GVHD, without increased risk of relapse or transplant related mortality. Severe CRS was not seen in this trial, and no anti-IL6 or steroid therapy was used. The addition of Jak inhibition to standard PtCy based GVHD prophylaxis was associated with encouraging rates of GRFS and OS on this pilot and expansion study. Patients on itacitinib have unique immune reconstitution characteristics both in terms of PBMC counts and signaling markers within subsets.

Cladribine Effects on T and B Cells and T Cell Reactivity in Multiple Sclerosis.

Cladribine tablet therapy is an efficacious treatment for multiple sclerosis (MS), however, its mechanism of action on T and B cell subsets remains unclear. The purpose of this study was to investigate the treatment effects of cladribine on the peripheral pool of T and B cells subsets and reactivity toward central nervous system (CNS) antigens. In this cross-sectional exploratory study, frequencies and absolute counts of peripheral T and B cell subsets and B cell cytokine production from untreated patients with relapsing-remitting MS (RRMS) and patients treated with cladribine for 1 year were measured using flow cytometry. Autoreactivity was assessed using a FluoroSpot assay. We found that 1 year after initiation of cladribine treatment, a lower number of CD4+ T cells was persisting whereas CD19+ B cell counts were normalized compared to untreated patients with RRMS. Follicular helper T cells and their effecter subsets producing cytokines exerting distinct B cell helper activity were lower and, additionally, the peripheral B cell pool was skewed toward a naïve and anti-inflammatory phenotype. Finally, reactivity to the recently identified CNS-enriched autoantigen RAS guanyl-releasing protein 2 (RASGRP2), but not to myelin basic protein and myelin oligodendrocyte glycoprotein, was lower in cladribine-treated patients. Together, these investigations on T and B cell subsets suggest that cladribine treatment impairs the B-T cell crosstalk and reduces their ability to mediate pathogenic effector functions. This may result in specific reduction of autoreactivity to RASGRP2 which is expressed in B cells and brain tissue. ANN NEUROL 2023.

Determining the mechanistic basis for Irgm1 mediated control of Mycobacterium tuberculosisinfection.

Abstract Irgm1 is a 47kDa IFN-γ inducible GTPase that has been shown to be essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. It was originally shown that Irgm1 is recruited to Mtb containing phagosomes in infected macrophages where it was proposed to be involved in autophagy-mediated clearance of Mtb. However, we have previously discovered that autophagy is not required in macrophages to control Mtb replication. In addition, it has since been reported that Irgm1 does not colocalize with phagosomes containing Mycobacterium bovis BCG in IFN-γ treated cells, suggesting that Irgm1 must have non-autophagic roles in TB infection that require detailed investigation. Irgm1 −/−mice were shown to have higher levels of type I interferon signaling, as well as CD4 +T cell survival defect, which could explain the susceptibility to Mtb infection, but this has yet to be determined. We infected Irgm1−/−mice with Mtb and found that they succumbed early to infection with higher bacterial burden, higher numbers of neutrophils, higher numbers of CD19 +B cells, and lower numbers of CD4 +T-lymphocytes in the lungs compared to wild-type (WT) mice. In addition, we found significantly increased expression of interferon-stimulated genes (ISGs) during Mtb infection of Irgm1−/−mice compared to WT. We also observed that T cell proliferation and lymph node responses were severely defective following Mtb infection in Irgm1−/−mice. Bone marrow chimera and transfer of Mtb-specific activated T lymphocytes into Irgm1−/−mice reverses the susceptibility to Mtb infection, indicating that a defect in T cell responses contributes to the susceptibility of Irgm1−/−mice to Mtb infection. This work was supported by Stephen I. Morse fellowship to Sumanta Kumar Naik and the Project supported by NIH AI142784 and AI132697 and BWF PATH Award to Dr. Christina Stallings.

Abstract 69: Minimally invasive brain metastases are characterized by elevated immune cell infiltrate

Abstract Background: Cancer metastasis to the brain is a common complication of advanced disease with limited therapeutic options. Inefficient treatment is influenced, in part, by the unique composition of the brain microenvironment. Brain metastases (BrM) grow in two distinct patterns, either as minimally invasive (MI) masses with well-defined borders, or as tumors with highly invasive (HI) growth into surrounding brain tissue. HI BrM are associated with poor prognoses compared to MI BrM; however, differences in the tumor immune microenvironments (TIME) between these two lesion types remain largely unknown. Here, we investigate how the TIME differs between HI and MI BrM. Methods: We use Nanostring Digital Spatial Profiling coupled with the Cancer Transcriptome Atlas panel on 5 MI and 15 HI BrM patient samples (lung and breast cancer). This technique enables specific isolation of cancer cells at the tumor-brain interface and quantification of 1,825 cancer-specific RNA targets. Additionally, we perform imaging mass cytometry (IMC) on 119 BrM samples (lung cancer, breast cancer, melanoma, other) from 46 patients, encompassing over 350,000 cells. Samples represent BrM from various primary sites, and include patient-matched samples from the brain-tumor interface (‘margin’) or the centre of the metastatic lesion (‘core’). Results: The Nanostring Digital Spatial Profiling revealed a list of 106 and 73 differentially expressed genes in MI vs HI breast and lung BrM, respectively. Gene set enrichment analyses revealed that an interferon gamma (IFNγ) pathway signature was enriched in MI BrM and lost in HI BrM, which was confirmed by immunohistochemical staining for pSTAT1, consistent with an “immune hot” TIME in MI BrM when compared to HI lesions. Using IMC technology, we identified 20 different cell types, activation states, and spatially-defined cellular neighbourhoods across our BrM patient samples. In comparison to MI samples, HI BrM have lower numbers of B cells, CD4+ T cells, and CD4- CD8- T cells in both the core and margin samples, and lower numbers of CD8+ T cells and regulatory T cells in the margin samples only. Discussion: These data suggest that HI BrM invade into an immunosuppressed microenvironment while MI BrM are characterized by an active anti-tumor immune infiltrate. Together, this work suggests potential immune regulation of BrM invasion, which warrants further investigation. Citation Format: Sarah M. Maritan, Elham Karimi, Matthew Dankner, Miranda W. Yu, Aldo Hernandez-Corchado, Morteza Rezanejad, Parvaneh Fallah, Benoit Fiset, Yuhong Wei, Stephanie Lam, Ali Nehme, Ian R. Watson, Morag Park, Yasser Riazalhosseini, Hamed Najafabadi, Kevin Petrecca, Marie-Christine Guiot, Daniela F. Quail, Logan A. Walsh, Peter M. Siegel. Minimally invasive brain metastases are characterized by elevated immune cell infiltrate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 69.

Abstract 5874: HSF1 downregulation of CCL5 reduces CD8 T cell trafficking in breast cancer

Abstract Breast cancer is the leading cause of cancer related deaths in women. The presence of cytotoxic immune cells, specifically CD8+ T cells, in breast tumors is associated with better patient outcomes. Our goal is to understand mechanisms that regulate the infiltration of CD8+ T cells. This will in turn, improve the treatment and enhance patient overall survival. Utilizing a novel HSF1 activity gene signature, we observed that HSF1 activity was negatively associated with the presence of CD8+ T cells in breast cancer patients. Both CIBERSORT analysis of TCGA data as well as direct assessment of patient primary specimens demonstrated that patient tumors with high HSF1 activity had lower numbers of CD8+ T cells. To functionally test this relationship, HSF1 was knocked down in 4T1 breast cancer cells and grown orthotopically in BALB/c mice. Tumors with HSF1 knockdown showed lower tumor volumes and increased CD8+ T cell infiltration. We subjected the control and knockdown tumors to single- cell RNA sequencing analysis and found increased immune cell presence specifically CD8+ T cell presence in knockdown tumors. We further tested the effect of CD8+ T cells on the growth of HSF1 knockdown tumors by depleting CD8+ T cells in BALB/c mice. HSF1 knockdown tumors were significantly larger with CD8+ T cell depletion suggesting a functional role for HSF1 to inhibit CD8+ T cell infiltration and protect the tumor from immune-mediated killing. To investigate the mechanism by which HSF1 affects CD8+ T cells, we investigated whether HSF1 affects chemotactic cytokines. We found that loss of HSF1 significantly increased secretion of CCL5, a known chemoattractant for CD8+ T cells. Loss of HSF1 also increased mRNA levels of CCL5, suggesting a transcriptional effect on CCL5. To test the importance of CCL5 in the phenotype of HSF1 knockdown, a transwell migration assay was performed wherein T cells from BALB/c mice were placed in the upper chamber of a transwell chamber and the lower chamber contained conditioned medium from 4T1 cells with knockdown of HSF1 with or without knockdown of CCL5. The lower chamber was subjected to flow cytometry for CD3/CD8 to identify CD8+ T cells and we observed that loss of HSF1 increased migration of CD8+ T cells and this was ablated with additional loss of CCL5. These results suggest CCL5 is a significant mediator for the recruitment of CD8+ T cells when HSF1 is lost in cancer cells. We propose the model whereby high HSF1 activity in breast cancer cells suppresses expression and secretion of the chemo-attractant cytokine CCL5 that ultimately leads to a decrease in CD8+ T cells in breast tumors microenvironment. Citation Format: Curteisha LeeAnn Jacobs, Sakhi Shah, Wen-Cheng Lu, Richard Carpenter, Haimanti Ray, Xin Lu, Sha Cao. HSF1 downregulation of CCL5 reduces CD8 T cell trafficking in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5874.

Leptomeningeal Disease (LMD) in Patients with Melanoma Metastases.

Leptomeningeal disease (LMD) is a devastating complication caused by seeding malignant cells to the cerebrospinal fluid (CSF) and the leptomeningeal membrane. LMD is diagnosed in 5-15% of patients with systemic malignancy. Management of LMD is challenging due to the biological and metabolic tumor microenvironment of LMD being largely unknown. Patients with LMD can present with a wide variety of signs and/or symptoms that could be multifocal and include headache, nausea, vomiting, diplopia, and weakness, among others. The median survival time for patients with LMD is measured in weeks and up to 3-6 months with aggressive management, and death usually occurs due to progressive neurologic dysfunction. In melanoma, LMD is associated with a suppressive immune microenvironment characterized by a high number of apoptotic and exhausted CD4+ T-cells, myeloid-derived suppressor cells, and a low number of CD8+ T-cells. Proteomics analysis revealed enrichment of complement cascade, which may disrupt the blood-CSF barrier. Clinical management of melanoma LMD consists primarily of radiation therapy, BRAF/MEK inhibitors as targeted therapy, and immunotherapy with anti-PD-1, anti-CTLA-4, and anti-LAG-3 immune checkpoint inhibitors. This review summarizes the biology and anatomic features of melanoma LMD, as well as the current therapeutic approaches.

Cytomegalovirus and Epstein-Barr virus infection during daratumumab treatment in patients with multiple myeloma

ABSTRACTS We explored the incidence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections in 131 patients with multiple myeloma (MM), 53 of whom received daratumumab (Dara) treatments. The Dara group had more RRMM patients than the group without Dara. CMV infection was significantly more common in patients treated with Dara (16.98%) than in patients treated with regimens without Dara (2.56%). During Dara treatments, 24.53% of patients developed CMV and/or EBV infections. Patients who developed infections had significantly lower levels of albumin and lymphocytes in their peripheral blood. The median time from the first Dara infusion to infection was 27 days. We observed NK cell depletion and T cell expansion during Dara-treatment. Patients with CMV and/or EBV infections had significantly lower numbers of NK cells, total T cells, and CD8 + T cells at 1 month, and lower numbers of CD8 + T cells at 2 months after the first Dara infusion than those without infections.

Association between immunity and viral shedding duration in non-severe SARS-CoV-2 Omicron variant-infected patients.

Coronavirus disease 2019 (COVID-19) is a respiratory-related disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 200 countries worldwide are affected by this disease. The Omicron variant of SARS-CoV-2 is the major epidemic variant worldwide and is characterized by higher infectivity. However, the immunity and risk factors for prolonged viral elimination in patients with non-severe SARS-CoV-2 Omicron variant infections are unclear. Therefore, this study aimed to examine the relationship between immunity and duration of viral elimination in non-severe SARS-CoV-2 Omicron variant-infected patients in Shanghai. In total, 108 non-severe SARS-CoV-2 Omicron variant-infected patients from Shanghai New International Expo Center Fangcang Shelter Hospital were recruited in this study. They were further allocated to the early elimination (EE) and prolonged elimination (PE) groups according to SARS-CoV-2 nucleic acid positivity duration. Compared to patients with EE, those with PE had increased serum concentrations of interleukin (IL)-5, IL-6, and IL-8; higher neutrophil count and neutrophil-to-lymphocyte ratio (NLR); lower lymphocyte, eosinophil, and red blood cell counts; and lower concentrations of hemoglobin and albumin (ALB). In lymphocyte subpopulation analysis, lower numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells and a higher CD4/CD8 ratio were observed in patients with PE. In addition, correlation analysis results revealed that cycle threshold values of SARS-CoV-2 Omicron variant ORF1ab and N were negatively correlated with IL-6 and IL-8 levels and positively correlated with eosinophil count in patients with COVID-19. Finally, multivariate regression analysis showed that ALB, CD4/CD8 ratio, NLR, and eosinophil count were predictors of the SARS-CoV-2 Omicron variant elimination. In this study, we identified that the ALB, CD4/CD8 ratio, NLR, and eosinophil count were risk factors for prolonged viral elimination in non-severe SARS-CoV-2 Omicron variant-infected patients. These factors might be efficient indicators in the diagnosis, evaluation, and prognosis monitoring of the disease.

Hematological and immune disorders in colorectal cancer patients with liver metastases after radiofrequency ablation

Background. Colorectal cancer (CRC) is one of the most common malignant neoplasms in the world. It ranks third in the structure of oncological morbidity and second in the structure of mortality. The liver is the most common site of metastatic spread of CR and 14 to 18% of patients have liver metastases at diagnosis. Radiofrequency ablation is currently the most promising method of treating metastatic lesions.&#x0D; Рurpose – to study quantitative changes in hematoimmunological indicators and their influence on antitumor reactivity in patients with colorectal cancer after RFA of liver metastases.&#x0D; Materials and Methods. Clinical and laboratory examination was carried out in 12 patients with colorectal cancer with metastases in the liver, the majority of patients were over 60 years old. Adenocarcinoma was histologically determined in all patients, and most of them had a moderate degree of malignancy (G2). The study was carried out in three stages: I – one day before radiofrequency ablation (RFA), II – 3 days after RFA, III – 14 days after RFA of liver metastases.&#x0D; Results. Immune and hematological indicators of the development of the inflammatory response after RFA in patients with colorectal cancer with liver metastases were established. On the 3rd day (II stage) after RFA, a significant increase in the total number of leukocytes, the ratio of neutrophils to lymphocytes, the number of eosinophils, a violation of the balance of CD4+ and CD8+ lymphocytes, a decrease in the number of NK- and NKT-cells, an increase in the percentage of CD3+ HLA-Dr -, CD4 +PD1+, CD8+PD1+ lymphocytes. On the 14th day (stage III), most of the parameters were close to those determined before the treatment, except for the reduced number of NK and NKT cells and the increase in the level of platelets.&#x0D; Conclusions. A number of hematological changes on the 3rd day after RFA related to the inflammatory reaction were identified: an increase in the total number of leukocytes, an increase in the ratio of neutrophils/lymphocytes from 1.72 to 4.12, a significant decrease in the relative and absolute number of eosinophils. But on the 14th day, after the inflammatory reaction subsided, the normalization of most of the studied indicators was observed. A violation of the subpopulation composition of lymphocytes was established in patients with CR metastases in the liver, on the 3rd day after RFA. Those that had taken place even before the intervention (low number of CD8+ lymphocytes and NK cells) and additional ones appeared (increased CD4+/CD8+ ratio, increased percentage of CD3+ cells HLA-Dr+, CD4+ and CD8+ lymphocytes PD1+ (CD279+), decrease in the number of NKT cells). On the 14th day, the picture approached the initial one, with the exception of the number of NK and NKT cells. We believe that timely correction of inflammatory immunosuppression in the early days after RFA can shorten the period of vulnerability to recurrence of CR, and in the long term potentiate the positive effect of RFA on antitumor reactivity.

The State of Cellular Immunity in Children with Infectious Mononucleosis

The main feature of infectious mononucleosis in children is the defeat of the immune system, primarily the T-cell link. Undoubtedly, the low number of CD4 + lymphocytes is associated with reduced resistance to secondary infections. This requires an inadequate immunological response and leads to the chronicity of infectious processes.

Ex Vivo Culture with Albumin Greatly Increases the Immunosuppressive Subset of T Lymphocytes

Emerging studies suggest that the expression of co-inhibitory receptors such as PD1, TIM3, TIGIT, and LAG3 on the T cell surface is important in regulating T cell response. However, the low number of cells expressing such co-inhibitory receptors in peripheral blood severely limits their clinical potential in treating GVHD. In this study, we introduce a novel method to culture and obtain a high number of CD3+PD1+TIM3+ lymphocytes that also exert strong immunosuppression. We demonstrate that culturing CD3+ cells isolated from G-CSF mobilized peripheral blood stem cells (G-PBSCs) with human serum albumin (HSA) greatly increases the number of immunosuppressive CD3+ cells. Also, we confirm that the CD3+PD1+TIM3+ lymphocytes are the most potent subset of the CD3+ cells that induce immunosuppression and inhibit T cell proliferation. Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3+ cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. Isolated CD3+ cells were cultured with 5% HSA for 4 days (1x105 cells/mL). PD1, TIM3, LAG3, and TIGIT expressions were analyzed using flow cytometry (FACS). CD3+, CD3+PD1+, and CD3+PD1+TIM3+ lymphocytes were sorted and cultured with irradiated allo-MNCs for three days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. Results. We discovered that treating G-PBSC cells with 5% HSA resulted in a dramatic increase in the number of immunosuppressive lymphocytes after the HSA treatment. In untreated G-PBSCs, there were very low numbers of CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ lymphocytes (0.9±0.3x103, 0.1±0.1x103, and 0.3±0.2x103, respectively). In G-PBSCs, the HSA treatment markedly increased the cell numbers (47.6±1.9x103, 7.8±3.0x103, and 0.7±0.3x103, respectively) while resulting in a mild increase in the total number of CD3+ cells (1.2-fold increase, 1x105 to 1.2±0.4x105). Our data also demonstrated significant percent changes in subpopulations of CD3+ lymphocytes. In untreated G-PBSCs, very low percentages of cells were CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ (9.0±3.1%, 1.2±0.1% and 3.2±2.8% in G-PBSC, respectively). After the HSA treatment, the percentages of the immunosuppressive lymphocytes in G-PBSCs markedly increased to 39.7±9.1%, 6.5±1.3%, and 0.6±0.4%, respectively. In particular, CD3+PD1+TIM3+ lymphocytes demonstrated the greatest increase in the cell number and the percentage after the HSA treatment, increasing by ~52 fold. Next, we tested whether the HSA-treatment induced proliferation of immunosuppressive T cells translated into increased immunosuppression. To do this, we used CSFE-MLR, co-cultured the CD3+ G-PBSCs with allo-MNCs, and analyzed the number of unstimulated T cells. The HSA-treated CD3+ G-PBSCs significantly increased the levels of unstimulated T cells compared to the untreated G-PBSCs (% of unstimulated T cells in untreated cells vs. in HSA-treated cells; 0.5±0.1 vs. 15.4±11.7), indicating that the HSA treatment increased CD3+ cell-mediated immunosuppression. Lastly, we tested the immunosuppressive effects of CD3+PD1+ and CD3+PD1-lymphocytes in HSA-treated CD3+ G-PBSCs. Both CD3+PD1+ and CD3+PD1+TIM3+ lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3+PD1-lymphocytes and CD3+PD1+TIM3- lymphocytes, respectively (% of unstimulated T cells in CD3+PD1+ vs. in CD3+PD1-lymphocytes, 27.9±5.5 vs. 4.3±7.5, and % of unstimulated T cells in CD3+PD1+TIM3+ vs. in CD3+PD1+TIM3- lymphocytes, 46.1±5.5 vs. 17.4±7.4). Collectively, these data suggest that PD1 and TIM3 are key markers in the HSA-dependent increase in immunosuppression. Conclusion. The ex vivo culture of CD3+ G-PBSCs with HSA markedly increases both the number and the effect of immunosuppressive CD3+ lymphocytes. Our analysis also indicates that the increased immunosuppression is likely due to the proliferation of CD3+PD1+TIM3+ lymphocytes. Taken together, our data suggest that the ex vivo HSA treatment of CD3+ G-PBSCs shows promising therapeutic potential for treating patients with GVHD.

Induction of pulmonary HLA-G expression by SARS-CoV-2 infection

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal–fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

Correlation study of PD-L1, CD4, CD8, and PD-1 in primary diffuse large B-cell lymphoma of the central nervous system.

We investigated the clinicopathological role of the PD-1/PD-L1 pathway in the primary diffuse large B-cell lymphoma of the central nervous system (PCNS-DLBCL). Standardized staining for PD-L1 was performed by machine staining, and internationally accepted interpretation methods were used. The PD-L1 immunostaining ≥20% of all cells in slices was defined as high expression of PD-L1. CD4, CD8, and PD-1 tumor-infiltrating lymphocytes (TILs) were enumerated, and the median was defined as the cutoff value. Values higher than the median was defined as high expression. Thirty-four cases (64.2%) showed high expression of PD-L1. PD-L1 expression was associated with a good prognosis when 20% was considered as cutoff value and had the smallest P value. By contrast, a low number of CD8+ or PD-1+ TILs was associated with poor prognosis. Patients with low expression of PD-L1 had poor overall survival (P=0.001), and those with increased CD8 or PD-1 TILs tended to have improved overall survival (P=0.004 and 0.024, respectively). Low number of monocytes, increased number of lymphocytes, IPI score ≥2, ECOG PS ≥2, LDH ≥250, and Ki67≥70% were independent prognostic factors for OS. In conclusion, PD-L1 expression, CD8 and PD-1 TILs, monocyte status, and ECOG PS might be prognostic markers and therapeutic targets of PCNS-DLBCL.