Off-Target HLA Cross-Reactivity By (Third Party) Virus-Specific T Cells Is Surprisingly Affected By HLA Restriction and HLA Background but Not By Virus Specificity

Reactivations of cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (AdV) occur frequently in immune compromised patients after allogeneic stem cell transplantation (alloSCT) and cause high morbidity and mortality. T-cell immunity is essential for anti-viral protection, but a fully competent T-cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a graft from a virus non-experienced donor are at risk. Adoptive transfer of partially HLA-matched virus-specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, such T cells harbor a risk of mediating off-target toxicity due to allo-HLA cross-reactivity. It is not currently known whether the degree of allo-HLA cross-reactivity is random or whether rules exist that might allow prediction of specific T-cell populations. Here, we investigated whether virus specificity, HLA type of the donor or HLA restriction of the virus-specific T cells influence the risk of allo-HLA cross-reactivity. Through cell sorting using tetramers for various peptides from CMV, EBV and AdV, 164 CD8 T-cell populations (21 specificities) were isolated from peripheral blood of 24 healthy donors, homozygous for HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. Forty percent of all virus-specific T-cell populations exerted allo-HLA cross-reactivity. Similar frequencies were found for the various viral specificities showing 33% of the CMV, 43% of the EBV and 38% of the AdV-specific T-cell populations to be allo-HLA cross-reactive. Surprisingly, a much larger fraction of the HLA-B*08:01-restricted virus-specific T-cell populations exhibited allo-HLA cross-reactivity (77%) than from those restricted by the other HLAs (32% of HLA-A*01:01, 38% of HLA-A*02:01 and 26% of HLA-B*07:02-restricted virus-specific T-cell populations). HLA-B*08:01-restricted virus-specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 different allogeneic EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02-restricted virus-specific T cells reacted to a median of 1, 2 and 3 (range 1-7) different allogeneic EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivities using a panel of 40 different single HLA-A, B, or C-transduced K562 cells further illustrated recurrent recognition of a restricted group of allogeneic HLA-B molecules by HLA-B*08:01-restricted T-cell populations, mediated by single T-cell clones. Heterozygosity for recurrently recognized allo-HLA-B molecules led to a significant decrease in the broadness of allo-HLA cross-reactivity by HLA-B*08:01-restricted T-cell populations, presumably due to negative thymic selection. In contrast, heterozygosity HLA-B molecules that were not part of the restricted group of cross-recognized alleles did not significantly decrease allo-HLA cross-reactivity. These data show that allo-HLA cross-reactivity by virus-specific T cells is highly influenced by their HLA restriction and the HLA background of the donors, but not by their virus specificity. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus-specific T-cell populations isolated from homozygous donors, HLA-B*08:01-restricted virus-specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity with recurrent recognition of groups of specific mismatched allogeneic HLA-B alleles. Our results indicate that selection of virus-specific T cells with specific HLA restrictions and HLA backgrounds may decrease the risk of off-target toxicity after infusion of third-party virus-specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.

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The Scope of Allo-HLA Cross-Reactivity By (Third Party) Virus Specific T Cells Is Surprisingly Affected By HLA Restriction Rather Than Virus Specificity

Blood ◽

10.1182/blood-2018-99-116028 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2048-2048

Author(s):

Wesley Huisman ◽

Didier A.T. Leboux ◽

Lieve E. van der Maarel ◽

Lois Hageman ◽

Derk Amsen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

K562 Cells ◽

Cross Reactivity ◽

Third Party ◽

Cell Populations ◽

Hla Alleles ◽

Hla Restriction ◽

T Cell Lines

Abstract Reactivations of cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) are frequently seen in immune compromised patients after allogeneic stem cell transplantation (alloSCT), and are associated with high morbidity and mortality. T cell immunity is essential for anti-viral protection, but a fully competent T cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a virus non- experienced donor are at risk of developing severe complications. Adoptive transfer of partially HLA-matched virus specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, these partially HLA-matched T cells harbor a risk of mediating allo-HLA cross-reactivity. Here, we investigated whether virus specificity and HLA restriction of the virus specific T cells influence the risk of allo-HLA cross-reactivity, and thus the development of GVHD. To determine the occurrence and diversity of allo-HLA cross-reactivity, virus specific CD8 T cells from homozygous HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02 donors were isolated by cell sorting using tetramers for various peptides from CMV, EBV and AdV. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. A significant proportion of the virus specific T cell populations (n=174; 20 specificities) isolated from 27 healthy donors exerted allo-HLA cross-reactivity, as measured by recognition of 1 or more HLA mismatched EBV-LCLs from the panel. Similar frequencies were found for the various viral specificities showing 30% of the CMV, 46% of the EBV and 36% of the AdV-specific T cell populations to be allo-HLA cross-reactive. However, for some specificities (e.g. HLA-A*0201-restricted EBV-LMP2-FLY) allo-HLA cross-reactivity was infrequent (n=1/11), whereas for other specificities (e.g. HLA-B*08:01-restricted EBV-BZLF1-RAK) the majority of the T cell populations (n=9/13) was allo-HLA reactive. Surprisingly, a much larger fraction of HLA-B*08:01 restricted virus specific T cell populations showed allo-HLA cross-reactivity (72%, 36 out of 50 T cell lines), compared to the other HLA restricted virus specific T cell populations (29% of HLA-A*01:01, 30% of HLA-A*02:01 and 26% of HLA-B*07:02 restricted virus specific T cell lines). HLA-B*08:01 restricted virus specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 allo EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02 restricted virus specific T cells reacted to a median of 1, 2 and 3 (ranges 1-7) allo EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivity using the panel of 40 different single HLA transduced K562 cells further illustrated the extensive allo-HLA cross-reactivity for HLA-B*08:01 restricted T cells isolated from homozygous HLA-A*01/B*08 donors compared to virus specific T cells restricted by other HLA alleles. These data show that allo-HLA cross-reactivity by virus specific T cells is highly influenced by the HLA restriction and not by the viral specificity of the T cell populations. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus specific T cell populations isolated from homozygous donors, HLA-B*08:01 restricted virus specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity. Our results indicate that selection of virus specific T cells with specific HLA restrictions may decrease the risk of developing GVHD after infusion of third-party virus specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.

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Magnitude of Off-Target Allo-HLA Reactivity by Third-Party Donor-Derived Virus-Specific T Cells Is Dictated by HLA-Restriction

Frontiers in Immunology ◽

10.3389/fimmu.2021.630440 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wesley Huisman ◽

Didier A. T. Leboux ◽

Lieve E. van der Maarel ◽

Lois Hageman ◽

Derk Amsen ◽

...

Keyword(s):

T Cells ◽

Hla Class I ◽

K562 Cells ◽

Cross Reactivity ◽

Third Party ◽

Thymic Selection ◽

Hla Restriction ◽

Selection For ◽

Hla Molecules ◽

Selection Of

T-cell products derived from third-party donors are clinically applied, but harbor the risk of off-target toxicity via induction of allo-HLA cross-reactivity directed against mismatched alleles. We used third-party donor-derived virus-specific T cells as model to investigate whether virus-specificity, HLA restriction and/or HLA background can predict the risk of allo-HLA cross-reactivity. Virus-specific CD8pos T cells were isolated from HLA-A*01:01/B*08:01 or HLA-A*02:01/B*07:02 positive donors. Allo-HLA cross-reactivity was tested using an EBV-LCL panel covering 116 allogeneic HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA-class-I alleles of interest. HLA-B*08:01-restricted T cells showed the highest frequency and diversity of allo-HLA cross-reactivity, regardless of virus-specificity, which was skewed toward multiple recurrent allogeneic HLA-B molecules. Thymic selection for other HLA-B alleles significantly influenced the level of allo-HLA cross-reactivity mediated by HLA-B*08:01-restricted T cells. These results suggest that the degree and specificity of allo-HLA cross-reactivity by T cells follow rules. The risk of off-target toxicity after infusion of incompletely matched third-party donor-derived virus-specific T cells may be reduced by selection of T cells with a specific HLA restriction and background.

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Therapeutic Infusion of Partially HLA-Matched Third-Party Virus-Specific T Cells in HSCT Patients with Refractory Viral Infection

Blood ◽

10.1182/blood.v124.21.3835.3835 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3835-3835

Author(s):

Leighton Edward Clancy ◽

Emily Blyth ◽

Barbara Withers ◽

Jane Burgess ◽

Renee Simms ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Third Party ◽

Viral Reactivation ◽

Antigen Specificity ◽

Specific Product ◽

Allogeneic Hsct ◽

Mhc Multimer

Abstract Introduction Adoptive transfer of donor derived virus specific T cells (VST) can be effective therapy for infections in allogeneic HSCT recipients. However, this is not a practical strategy to treat acute infections due to the time required to prepare products and potential unavailability of transplant donors. To overcome this, treatment with cryopreserved partially HLA-matched T cells from third-party donors is being investigated. A recent report described disease resolution using cells matched at only one or two HLA alleles (Leen et al., (2013) Blood 121(26):5113-23). This less stringent requirement for matching would allow a small bank of cells to provide most patients with a therapeutic product. We describe the establishment of a virus specific T cell bank in Australia with centralized manufacturing by the Westmead Hospital BMT laboratory. The bank has been used to treat patients in multiple states in a Phase I clinical trial to treat patients who have failed antiviral pharmacotherapy. Aim To assess the safety and feasibility of treatment with partially HLA-matched VSTs derived from third-party donors for refractory cytomegalovirus (CMV), Epstein-Barr Virus (EBV), or adenoviral (AdV) infection in allogeneic HSCT patients. Methods We generated a bank of cryopreserved VSTs from peripheral blood or G-CSF mobilized stem cell product of healthy donors. Products were generated by co-culturing PBMC with dendritic cells loaded with overlapping peptides covering CMV pp65, AdV hexon or EBV BZLF1, LMP2A and EBNA1 proteins. Cultures were re-stimulated once with peptide loaded DC and cultured for 14 days with IL-2. Products were assessed for phenotype, sterility and specificity by MHC multimer staining where applicable or production of interferon-gamma in response to peptides by flow cytometry. Patients with persistent viral reactivation/infection after 2 weeks of standard therapy were eligible to receive up to 4 fortnightly infusions of 2x107 cells/m2partially HLA matched (minimum 1/6) CMV, EBV, or AdV specific T cells, and were followed for up to 12 months. Results T cell products were expanded from 25 donors to create a bank of 177 bags of VSTs (75 CMV, 47 AdV and 55 EBV). CMV specific products were predominantly T cells (mean 95.8±3%) with a higher proportion of CD8+ compared to CD4+ T cells (mean 66.6±23.9% versus 20.1±6.2%). Specificity was mapped by MHC multimer staining to epitopes restricted to common HLA types including HLA-A*0101, HLA-A*0201, HLA-A*2402, HLA-B*0702 and HLA-B*3501. AdV specific T cells had a higher proportion of CD4+ T cells (mean 64.6±23.8% versus 34.2±20.1% CD8 T cells). Specificity was mapped to CD8 epitopes restricted to HLA-A*0101 and HLA-A*2402 as well as 10 CD4 T cell epitopes restricted to three HLA-DRB1 alleles (DRB1*0301, DRB1*0701, DRB1*1501). EBV specific products contained a mix of CD8+ and CD4+T cells (mean 38.9%±18% AND 42.5±23.1% respectively). The antigen specificity of EBV products showed high variability between donors. Dominant responses to known MHC class I restricted epitopes were infrequent though responses were mapped to HLA-A*0201 and HLA-A*2402 restricted LMP2A epitopes, a HLA-B*0801 restricted BZLF1 epitope and a HLA-B*0702 restricted EBNA1 epitope. Based on HLA frequency analysis in the Australian recipient population we estimate 94%, 89% and 74% of patients would have access to a CMV, AdV and EBV specific product respectively with the current bank. To date nine patients have received VSTs, with median follow-up of 5.5 months (0-12 months). All patients had treatment resistant CMV after a median of 26 days (19-116 days) prior therapy. Six patients received a single infusion and 3 patients received 2, 3 and 4 infusions respectively. HLA matching ranged from 2-4/6 HLA match. There were no instances of 24hr infusion related toxicity. Follow-up data is available for 7 patients. One patient with chronic hepatitis C developed abnormal liver function tests 3 months post-infusion. One patient died from presumed progressive CMV disease 6 months post-enrolment. Five patients achieved a best response of CMV PCR negativity (2 with complete resolution of CMV-colitis). One patient has shown >50% reduction in CMV copy number over 3 weeks. Conclusion The infusion of third party CMV specific T cells is a promising therapy that offers the advantage of rapid availability, centralized manufacturing and relatively low cost per dose when produced on a large scale. Disclosures No relevant conflicts of interest to declare.

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P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells

Blood ◽

10.1182/blood-2001-12-0353 ◽

2002 ◽

Vol 100 (2) ◽

pp. 375-382 ◽

Cited By ~ 63

Author(s):

Martin Guimond ◽

Antonia Balassy ◽

Mélanie Barrette ◽

Sylvie Brochu ◽

Claude Perreault ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Third Party ◽

Cell Populations ◽

Activated T Cells ◽

Graft Versus Host ◽

Histocompatibility Complex ◽

P Glycoprotein ◽

Selective Elimination

Abstract T lymphocytes have been found to harbor P-glycoprotein (Pgp) and to demonstrate modulation of its ion channel transporter function according to the state of activation of T lymphocytes. We hypothesized that cytotoxic chemicals that are extruded by Pgp could be used to specifically eliminate immunoreactive T-cell populations. In this study, we evaluated the capacity of 4,5-dibromorhodamine methyl ester (TH9402), a photosensitizer structurally similar to rhodamine, a dye transported by Pgp, and which becomes highly cytotoxic on activation with visible light to selectively deplete alloreactive T lymphocytes. Stimulation of T cells with mitogens or allogeneic major histocompatibility complex–mismatched cells resulted in the preferential retention of the TH9402 rhodamine-derivative in activated T cells, both CD4+ and CD8+. Photodynamic cell therapy of TH9402-exposed T cells led to the selective elimination of immunoreactive T-cell populations. In addition, this treatment preserved resting T cells and their capacity to respond to third-party cells. Inhibition of Pgp enhanced cellular trapping of the dye in nonactivated T cells and resulted in their depletion after exposure to light. Targeting of Pgp-deficient cells may therefore represent an appealing strategy for the prevention and treatment of graft-versus-host disease and other alloimmune or autoimmune disorders.

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Isolation and Validation of the First Functional HLA-a*01:01 Restricted EBV-LMP2 Specific T Cells for Treatment of EBV Associated Type II/III Lymphomas

Blood ◽

10.1182/blood-2018-99-116127 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1578-1578

Author(s):

Wesley Huisman ◽

Lieve E. van der Maarel ◽

Lois Hageman ◽

Rob C.M. de Jong ◽

Derk Amsen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

K562 Cells ◽

Nuclear Antigen ◽

Cell Populations ◽

High Avidity ◽

Type Ii ◽

Functional Avidity ◽

Specific Peptide

Abstract Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the virus. Although many EBV associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Different strategies have been developed to manufacture EBV LMP1/2 specific T cell products for clinical application. Surprisingly, to date, no EBV specific T cells recognizing a peptide in the common HLA allele A*01:01 have been found. Furthermore, an HLA-A*01:01 associated risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported. A need thus exists for HLA-A*01:01 restricted EBV specific T cell products, especially EBV-LMP1/2 specific T cells. Based on MHC class I peptide predictions, HLA-A*01:01 binding peptides derived from different immunogenic EBV antigens were identified, and HLA-A*01:01/peptide tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). For these 5 specificities, tetramer positive CD8 T cells were sorted by flow cytometry from peripheral blood mononuclear cells (PBMC) of 6 HLA-A*01:01 positive healthy donors and subsequently cultured with 1µM of specific peptide. Specificity of expanded T cells was confirmed by tetramer staining. Functional avidity was assessed using TAP2 deficient T2 cells transduced with HLA-A*01:01 and exogenously loaded with 10-12M to 10-6M of the respective specific peptide. The recognition of endogenously processed and presented antigen was analyzed using K562 cells transduced with HLA-A*01:01 and a retroviral vector encoding the full protein sequence of LMP2, HLA-A*01:01 positive EBV-LCLs and IFNy ELISA as read-out. HLA-A*01:01 restricted EBV specific T cells were present at low frequencies in total PBMCs from all 6 donors (EBNA3A-YTD 0.008%; range 0.001-0.02%, EBNA3A-FLQ 0.01%; range 0.0016-0.012%, BZLF1-FTP 0.0033%; range 0.0025-0.0075%, LMP2-ESE 0.0049%; range 0.0015-0.15% and LMP2-LTE 0.003%; range 0.002-0.025%). After flow cytometric cell sorting, only EBV-LMP2-ESE specific T cells of 5/6 donors expanded, resulting in pure tetramer+ CD8 T cell lines. Analysis of the T cell receptor beta chain (TCR-Vβ) usage showed that these EBV-LMP2-ESE specific T cell populations were oligoclonal. To further analyze their functional avidity, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01 transduced TAP2 deficient T2 cells exogenously loaded with peptide. Four out of 5 isolated T cell populations recognized T2 cells loaded with 1-103nM peptide, indicating intermediate to high avidity. To investigate whether this specific LMP2-derived peptide can be functionally processed and presented and whether these T cells were able to recognize endogenously processed and presented antigen, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01/LMP2 transduced K562 cells. All functional EBV-LMP2-ESE specific T cell populations could recognize these transduced K562 cell lines, indicating that this LMP2-ESE peptide is successfully processed, presented and recognized by EBV-LMP2-ESE specific T cells. Recognition of EBV-LCLs illustrated the potential of the EBV-LMP2-ESE specific T cells to recognize endogenous LMP2. We describe the isolation and validation of the first functional HLA-A*01:01 restricted EBV-LMP2 specific T-cell populations, which can be used for adoptive transfer to treat EBV associated type II/III lymphomas, malignancies of epithelial origin and PTLDs. Despite the very low frequency of these T cells, we were able to obtain these T cells from 5 out of 6 donors and showed their capacity to recognize both exogenously loaded as endogenously processed and presented EBV-LMP2-ESE peptide. Disclosures No relevant conflicts of interest to declare.

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Suppressor T-cell mechanisms in contact sensitivity. III. Apparent non-major histocompatibility complex restriction is a result of multiple sets of major histocompatibility complex-specific suppressor T cells induced by syngeneic 2,4-dinitrophenyl-modified lymphoid cells.

Journal of Experimental Medicine ◽

10.1084/jem.150.3.676 ◽

1979 ◽

Vol 150 (3) ◽

pp. 676-692 ◽

Cited By ~ 6

Author(s):

S D Miller

Keyword(s):

T Cells ◽

Major Histocompatibility Complex ◽

T Cell ◽

Cross Reactivity ◽

Lymphoid Cells ◽

Third Party ◽

Contact Sensitivity ◽

Major Histocompatibility ◽

Suppressor T Cells ◽

Histocompatibility Complex

This report has examined the mechanisms by which major histocompatibility complex (MHC) non-restricted suppressor T cells (Ts), induced by the i.v. injection of 2,4-dinitropheny (DNP)-modified, syngeneic lymphoid cells (DNP-LC), suppress the passive transfer of contact sensitivity mediated by syngeneic and allogeneic immune delayed hypersensitivity T cells (TDH). In terms of suppression of syngeneic TDH, it was found that the suppressive action of the Ts was only blocked by pretreatment with soluble syngeneic DNP-LC membrane preparations. Monomeric DNP-lysine, polymeric DNP-protein conjugates, and syngeneic TNP-LC membranes did not inhibit Ts function. Further experiments showed that inhibition of syngeneic suppression could be achieved by DNP-modified-membrane preparations that were only H-2D-region compatible with the Ts donor. Thus, Ts antigen receptors in this system specifically recognize DNP-modified H-2D-region determinants. In contrast, it was found that pretreatment os syninduced Ts with syngeneic DNP-LC membranes did not inhibit the ability to suppress allogeneic TDH. However, pretreatment of Ts with DNP-allogeneic membranes which were H-2D-end compatible to the allogeneic target TDH eliminated their ability to suppress the specific allogeneic TDH, leaving intact suppression of syngeneic or third party TDH. It is proposed that perturbation of the immune system by i.v. injection of syngeneic NDP-LC leads to the induction of a polyclonal wave of DNP-specific Ts activity. Some members of this set of Ts recognize DNP-self MHC determinants with moderate affinity and are thus specifically inhibited after pretreatment with those DNP-self determinants. Other members of this set display receptors which cross-react with high affinity with DNP-allogeneic determinants and thus suppress allogeneic TDH cells. These allosuppressive clones can thus be specifically inhibited only by pretreatment with DNP-LC membranes, MHC-compatible with the target TDH. The data are discussed in terms of current models of T-cell cross-reactivity and T-cell-receptor recognition.

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Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

Scientific Reports ◽

10.1038/s41598-021-81474-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Josephine F. Reijneveld ◽

Mira Holzheimer ◽

David C. Young ◽

Kattya Lopez ◽

Sara Suliman ◽

...

Keyword(s):

T Cells ◽

Fatty Acid ◽

Immune System ◽

T Cell ◽

Cross Reactivity ◽

Lipid Binding ◽

Human Immune System ◽

Human T Cell ◽

Human T Cells ◽

Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

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49 In situ phenotypic analysis of T cells in the tumor microenvironment of a pre-clinical model of non-small cell lung cancer (NSCLC) by tissue sectioning and whole-mount immunofluorescence imaging

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0049 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A52-A52

Author(s):

Elen Torres ◽

Stefani Spranger

Keyword(s):

T Cells ◽

Spatial Distribution ◽

T Cell ◽

Spatial Location ◽

Cell Infiltration ◽

Cell Populations ◽

Tumor Bearing ◽

T Cell Infiltration ◽

Volume Imaging ◽

Whole Mount

BackgroundUnderstanding the interactions between tumor and immune cells is critical for improving current immunotherapies. Pre-clinical and clinical evidence has shown that failed T cell infiltration into lung cancer lesions might be associated with low responsiveness towards checkpoint blockade.1 For this reason, it is necessary to characterize not only the phenotype of T cells in tumor-bearing lungs but also their spatial location in the tumor microenvironment (TME). Multiplex immunofluorescence staining allows the simultaneous use of several cell markers to study the state and the spatial location of cell populations in the tissue of interest. Although this technique is usually applied to thin tissue sections (5 to 12 µm), the analysis of large tissue volumes may provide a better understanding of the spatial distribution of cells in relation to the TME. Here, we analyzed the number and spatial distribution of cytotoxic T cells and other immune cells in the TME of tumor-bearing lungs, using both 12 µm sections and whole-mount preparations imaged by confocal microscopy.MethodsLung tumors were induced in C57BL/6 mice by tail vein injection of a cancer cell line derived from KrasG12D/+ and Tp53-/- mice. Lung tissue with a diverse degree of T cell infiltration was collected after 21 days post tumor induction. Tissue was fixed in 4% PFA, followed by snap-frozen for sectioning. Whole-mount preparations were processed according to Weizhe Li et al. (2019) 2 for tissue clearing and multiplex volume imaging. T cells were labeled with CD8 and FOXP3 antibodies to identify cytotoxic or regulatory T cells, respectively. Tumor cells were labeled with a pan-Keratin antibody. Images were acquired using a Leica SP8 confocal microscope. FIJI3 and IMARIS were used for image processing.ResultsWe identified both cytotoxic and regulatory T cell populations in the TME using thin sections and whole-mount. However, using whole-mount after tissue clearing allowed us to better evaluate the spatial distribution of the T cell populations in relation to the tumor structure. Furthermore, tissue clearance facilitates the imaging of larger volumes using multiplex immunofluorescence.ConclusionsAnalysis of large lung tissue volumes provides a better understanding of the location of immune cell populations in relation to the TME and allows to study heterogeneous immune infiltration on a per-lesion base. This valuable information will improve the characterization of the TME and the definition of cancer-immune phenotypes in NSCLC.ReferencesTeng MW, et al., Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res 2015;75(11): p. 2139–45.Li W, Germain RN, and Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 2019;14(6): p. 1708–1733.Schindelin J, et al, Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7): p. 676–82.

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CMVpp65-Specific T cells generated from naïve T cell populations recognize atypical but not canonical epitopes and may be protective in Vivo

Cytotherapy ◽

10.1016/j.jcyt.2015.03.315 ◽

2015 ◽

Vol 17 (6) ◽

pp. S9-S10

Author(s):

Patrick Hanley ◽

Joseph Melenhorst ◽

Russell Cruz ◽

Caridad Martinez ◽

Helen Heslop ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Populations ◽

Naïve T Cell ◽

Naive T Cell

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Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus

Journal of Virology ◽

10.1128/jvi.00302-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6566-6577 ◽

Cited By ~ 52

Author(s):

Katherine A. Richards ◽

Francisco A. Chaves ◽

Andrea J. Sant

Keyword(s):

T Cells ◽

Influenza Virus ◽

T Cell ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Cd4 T Cells ◽

Cross Reactivity ◽

Cd4 T Cell ◽

Ns1 Protein ◽

Infected Cells

ABSTRACT The specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.

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