Peripheral and systemic antigens elicit an expandable pool of resident memory CD8
            +
            T cells in the bone marrow

Abstract Multiple myeloma (MM) is a bone marrow (BM) resident hematological malignancy that is becoming increasingly recognized as one amenable to immunotherapy, although no therapies have yet provided durable, long-term disease control. Autologous stem cell transplantation (ASCT), the standard of care in eligible patients, provides a window for intervention with immunotherapy due to the induction of inflammation in the context of lymphodepletion at a time where there is also minimal residual disease and a disrupted tumor microenvironment (TME). We have previously established that the addition of T cells to BM grafts results in enhanced long-term myeloma control post-transplant in mice. Novel approaches aimed at improving and/or expanding the endogenous T cell response early post-ASCT may therefore prove highly effective with the benefit of avoiding ex vivo processing associated with other cellular therapies. To explore this, we utilized the IL-2/IL-15 mimetic NL-201: a de novo cytokine mimetic that signals via the beta and gamma subunits of the IL-2 receptor without engaging IL-2Rα (CD25). In pre-clinical studies, NL-201 has demonstrated the ability to signal to effector CD4 and CD8 T cells while avoiding the toxicity usually associated with IL-2 signaling via IL-2Rα. We hypothesized that NL-201 would enhance control of myeloma progression by stimulating T cell proliferation and activation early post-ASCT. We transplanted lethally irradiated Vk*MYC myeloma-bearing B6 recipients with BM and T cells graft from B6 donors and administered NL-201 from D+7 to week 6 (225 μg/kg weekly I.P). NL-201 promoted potent anti-myeloma immunity that was dependent on CD4 and CD8 T cells, but not NK cells (median survival was 68 days for control mice, unreached at >120 days for NL-201 alone or with NK depletion, 86 days for NL-201 with CD8 depletion, and 74 days with CD4 depletion; PBS vs NL-201 p<0.01; PBS vs NL-201 + αNK1.1 p<0.01; NL-201 vs NL-201 + αCD4 or αCD8 p<0.05). To further elucidate potential mechanisms of action we harvested BM from PBS and NL-201-treated mice 2 days after the last dose was administered and performed comprehensive immunophenotyping with high parameter flow cytometry. We grouped recipients based on whether they had controlled myeloma (MM-controlled) or had active disease progression at the time of harvest (MM-relapsed) to reveal immunological phenotypes that were dependent and independent of myeloma in the TME. In these experiments, all NL-201-treated recipients had controlled myeloma at time of harvest. Mechanistically, NL-201 significantly expanded the total number of CD8 T cells in the BM compared to PBS-treated mice with controlled or relapsed MM (PBS-treated mean CD8 T cell number was 1.0 x 10 5/femur vs 7.7 x 10 5/femur in NL-201-treated mice) but did not impact CD8 T cell number in peripheral blood. Memory CD8 T cells (CD44+CD62L+) were preferentially expanded, while the frequency of exhausted CD8 T cells (TOX +PD-1 +TIGIT +CD39 +; T EX) was reduced in NL-201-treated mice compared to both PBS-treated MM-relapsed and MM-controlled mice (75% T EX in PBS MM-relapsed, 15% PBS MM-controlled, 2% in NL-201; p<0.001). Surprisingly, >80% of the memory CD8 T cells in NL-201-treated mice produced granzyme B compared to <10% in PBS-treated mice. Granzyme B production was also observed in conventional CD4 T cells in response to NL-201 treatment, and the frequency of regulatory T cells was reduced by 50% after NL-201 compared to PBS MM-controlled and MM-relapsed mice (p<0.001). NL-201 expanded bone marrow resident cytotoxic memory CD8 and CD4 T cells that are resistant to exhaustion, whilst reducing the frequency of regulatory T cells in the BM TME. Together, these data highlight the promising therapeutic potential of NL-201 in multiple myeloma and support testing NL-201 in clinical trials for the treatment of hematological malignancies. Disclosures Hill: NapaJen Pharma: Consultancy; Roche: Research Funding; Syndax Pharmaceuticals: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Applied Molecular Transport: Research Funding; Compass Therapeutics: Research Funding; NeoLeukin Therapeutics: Consultancy; Generon Corporation: Consultancy.

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Abstract P328: Regulation Of Monocyte Physiology By Il-4 Stimulated Memory Cd8+ T-cells

Circulation Research ◽

10.1161/res.129.suppl_1.p328 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Philip Broughton ◽

Yusra Zaidi ◽

Miguel Troncoso ◽

Kristine Y DeLeon-Pennell

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Scar Tissue ◽

Positive Control ◽

Macrophage Phenotype ◽

Memory Cd8 T Cells ◽

Monocyte Migration ◽

Cell Pellet ◽

And Migration

Following a myocardial infarction (MI) monocytes and T-cells begin to infiltrate into the ischemic area in effort to remove necrotic debris and initiate formation of scar tissue. Interleukin (IL)-4 has been linked to improved cardiac wound healing via alterations in both the macrophage and T-cell populations. The goal of this study was determine if proteins secreted by IL-4 stimulated CD8+ T-cells would regulate monocyte physiology that would ultimately improve cardiac healing after an MI. Isolated splenic naïve CD8+ T-cells from day 0 (no MI) mice (n=5/sex/stimulation)were cultured in RPMI with either 0.1% FBS (unstimulated) or 0.1% FBS+ IL-4. After 24 hours of stimulation, the cells and media were collected and separated by centrifugation. The cell pellet was stained and analyzed for markers of activation (CD44) and memory (CD27) by flowcytometry. Conditioned media (secretome) was collected for stimulation of bone marrow monocytes (n=4; females only). After stimulation with the secretome, monocytes were analyzed for viability, phagocytosis, and macrophage phenotype by flow cytometry. Migration of the monocytes after stimulation was also measured using electric cell-substrate impedance sensing (ECIS). After IL-4 stimulation, there was a shift from effector (CD44+ CD27-) to the memory phenotype (CD44+ CD27+; p<0.05 vs unstimulated cells). Interestingly, bone marrow monocyte viability was decreased by 15% when stimulated with the secretome of IL-4 treated CD8+ T-cells compared to unstimulated CD8+ T-cells (0.1%). Phagocytosis was slightly elevated though not significant (p=0.07) in monocytes that were stimulated with the secretome from the IL-4 group compared to the unstimulated CD8+ T-cells. No differences were found in expression of macrophage markers F4/80 (p=0.532) or M1 marker CD86 (p=0.471). The secretome of IL-4 stimulated T-cells increased monocyte migration after wounding similar to levels of the positive control (monocytes in 10% FBS only). The data collected showed that IL-4 stimulated CD8+ T-cells were able to upregulate memory marker CD27. These memory-like CD8+ T-cells initiated monocyte phagocytosis and migration but decreased monocyte viability suggesting that they may play a role in regulating macrophage biology post-MI.

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A CpG-Activated Whole-Cell Vaccine ‘Boost’ Enhances the Anti- Lymphoma Efficacy of Immunotransplant

Blood ◽

10.1182/blood.v112.11.3627.3627 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3627-3627

Author(s):

Joshua Brody ◽

Matthew J. Goldstein ◽

Ronald Levy

Keyword(s):

Bone Marrow ◽

T Cells ◽

Tumor Immunity ◽

Cd8 T Cells ◽

Tumor Challenge ◽

Myeloablative Conditioning ◽

Memory Cd8 T Cells ◽

Specific Memory ◽

Tumor Protection ◽

Proliferative Signal

Abstract BACKGROUND: Previously, we demonstrated that an anti-lymphoma vaccine was made more powerful by ‘immunotransplant’ of vaccine-primed donor splenocytes into lethally irradiated, syngeneic bone marrow transplant recipients. Immunotransplant increased the proportion of tumor-specific, memory CD8 T cells by almost ten-fold and cured tumors several times larger than what could be cured by vaccination alone. We demonstrated that this increased anti-tumor effect correlated with the ‘homeostatic proliferative’ signal that tumor-specific donor T cells receive in lymphodepleted recipients. If the ‘‘homeostatic proliferative’ signal could synergize with other T cell activating signals, it is possible that the anti-tumor effect could be even further enhanced. METHODS: We asked whether the simultaneous transmission of ‘signal 1+2’ (the TCR-recognized antigen + co-stimulation) along with the ‘homeostatic proliferative’ signal could further enhance the anti-tumor immunity induced by immunotransplant. To optimize ‘signal 1+2’, lymphoma cells were incubated with a TLR9 agonistic CpG oligodeoxynucleotide, which has been shown to upregulate both surface MHC (signal 1) and co-stimulatory molecule (signal 2) expression. This CpG-NHL was irradiated and administered as a vaccine boost along with the previously described immunotransplant maneuver (i.e. vaccine-primed donor splenocytes and bone marrow transferred into lymphodepleted, syngeneic recipients). RESULTS: CpG-NHL ‘boosting’ increased the proportion of tumor-specific, memory CD8 T cells approximately four fold. In tumor protection experiments, addition of CpG-NHL boost to lower doses of transferred, vaccine-primed splenocytes, increased the proportion of surviving recipients from 0% to 100% in both subcutaneous and systemic tumor challenge models. The CpG-NHL boosting effect was specific to the immunotransplant of vaccine-primed splenocytes and did not induce significant tumor protection in combination with ‘sham’ immunotransplant of normal donor splenocytes. Conversely, the CpG-mediated pre-activation was essential, as ‘boosting’ with non-treated, irradiated NHL cells induced inferior tumor protection. Our prior work demonstrated that myeloablative conditioning of immunotransplant recipients was necessary for significant tumor protection. As a primary goal of this work is the modeling of the immunotransplant system for clinical translation, we asked whether the addition of the CpG-NHL boost could allow non-myeloablative conditioning approaches to be effective in protecting immunotransplant recipients from tumor challenge. We demonstrated that both sub-lethal irradiation and chemotherapeutic conditioned recipients developed significant anti-tumor immunity only when the CpG-NHL boost was combined with immunotransplant. CONCLUSIONS: Several clinical trials using adoptive transfer of anti-tumor lymphocytes have demonstrated objective clinical responses in patients with lymphoma and solid tumors. Our work suggests that such clinical approaches could be enhanced by post-transfer vaccine boosting and also provides a basis to study the interaction of the ‘homeostatic proliferative’ signal with ‘signal 1+2’.

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Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients

Bone Marrow Is a Major Reservoir and Site of Recruitment for Central Memory CD8+ T Cells

Human Bone Marrow: A Reservoir for “Enhanced Effector Memory” CD8+T Cells with Potent Recall Function

Faculty Opinions recommendation of Neutrophils transport antigen from the dermis to the bone marrow, initiating a source of memory CD8+ T cells.

Human bone marrow contains a subset of quiescent early memory CD8+T cells characterized by high CD127 expression and efflux capacity

Commentary: Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription

Neural-Cadherin Influences the Homing of Terminally Differentiated Memory CD8 T Cells to the Lymph Nodes and Bone Marrow

The IL-2/IL-15 Mimetic NL-201 Prevents Myeloma Relapse after ASCT By Expanding Highly Cytolytic T Cells in the Bone Marrow That Are Resistant to Exhaustion

Abstract P328: Regulation Of Monocyte Physiology By Il-4 Stimulated Memory Cd8+ T-cells

A CpG-Activated Whole-Cell Vaccine ‘Boost’ Enhances the Anti- Lymphoma Efficacy of Immunotransplant