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. 2022 Aug 23;6(16):4859-4872.
doi: 10.1182/bloodadvances.2022007005.

Dominant epitopes presented by prevalent HLA alleles permit wide use of banked CMVpp65 T cells in adoptive therapy

Aisha N Hasan  1   2 , Ekaterina Doubrovina  1   2 , Rosa Sottile  3 , Susan Prockop  1   2 , Martin G Klatt  4   5 , Glenn Heller  6 , Annamalai Selvakumar  1   2 , Lorna Barnett  1   2 , Katharine C Hsu  3   7   8 , Richard J O'Reilly  1   3   7   8
Affiliations

Dominant epitopes presented by prevalent HLA alleles permit wide use of banked CMVpp65 T cells in adoptive therapy

Aisha N Hasan et al. Blood Adv. .

Abstract

We established and characterized a bank of 138 CMVpp65 peptide-specific T-cell (CMVpp65CTLs) lines from healthy marrow transplant donors who consented to their use for treatment of individuals other than their transplant recipient. CMVpp65CTL lines included 131 containing predominantly CD8+ T cells and 7 CD4+ T cells. CD8+ CMVpp65CTLs were specific for 1 to 3 epitopes each presented by one of only 34 of the 148 class I alleles in the bank. Similarly, the 7 predominantly CD4+ CMVpp65CTL lines were each specific for epitopes presented by 14 of 40 HLA DR alleles in the bank. Although the number of HLA alleles presenting CMV epitopes is low, their prevalence is high, permitting selection of CMVpp65CTLs restricted by an HLA allele shared by transplant recipient and hematopoietic cell transplant donor for >90% of an ethnogeographically diverse population of hematopoietic cell transplant recipients. Within individuals, responses to CMVpp65 peptides presented by different HLA alleles are hierarchical. Furthermore, within groups, epitopes presented by HLA B*07:02 and HLA A*02:01 consistently elicit immunodominant CMVpp65CTLs, irrespective of other HLA alleles inherited. All dominant CMVpp65CTLs exhibited HLA-restricted cytotoxicity against epitope loaded targets and usually cleared CMV infections. However, immunodominant CMVpp65CTLs responding to epitopes presented by certain HLA B*35 alleles were ineffective in lysing CMV-infected cells in vitro or controlling CMV infections post adoptive therapy. Analysis of the hierarchy of T-cell responses to CMVpp65, the HLA alleles presenting immunodominant CMVpp65 epitopes, and the responses they induce may lead to detailed algorithms for optimal choice of third-party CMVpp65CTLs for effective adoptive therapy.

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Figures

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Graphical abstract
Figure 1.
Figure 1.
Mapping of CMVpp65 peptide epitope specificity and HLA restriction of a representative CMVpp65CTL line. (A) IFN-γ responses of T cells against a matrix of CMVpp65 peptide subpools. CD8+ T cells of the CMVpp65CTL line respond selectively to subpools #10 and #19, which uniquely share the QAIRETVEL peptide. Furthermore, these CMVpp65CTLs selectively lyse phytohemagglutinin-stimulated blasts loaded with this peptide that share with the CMVpp65CTLs the HLA-B*3503 and not peptide-loaded phytohemagglutinin-stimulated blasts sharing other HLA alleles in the CMVpp65CTL genotype. This line also contains a nondominant population of IFN-γ+ CD8+ T cells specific for peptide 44 uniquely shared by subpools 8 and 16 that contains the QWKEPDVYYT epitope that can be presented by the donor’s HLA C0401 allele. However, these T cells were not cytotoxic against peptide-loaded targets sharing any single HLA allele, with donor preventing ascertainment of HLA restriction. Note that a low-level (11%) alloresponse to DQB10503 alone and not against the same cells loaded with CMVpp65 peptides was also seen. (B) This CMVpp65CTL line contains dominant CD4+ T cells that selectively generate IFN-γ in response to subpools 3 and 23, which uniquely share the LARNLVPMVATV peptide, and lyse targets loaded with the peptide that selectively share only the HLA-DRB*1-0402 allele with the CMVpp65CTLs. This line also contains 2 other CMVpp65CTL populations: a small population of CD8+ T cells restricted by HLA A2601 specific for SHIMLDVAF, a CMVpp65 peptide in 15-mer #72 shared by pools 12 and 18, and an IFN-γ+ cytolytic CD4+ T-cell population restricted by HLA DQB, 0301, specific for subpools 8 and 20 sharing the 15-mer #92: EHPTFTSQYRIQGKL. E:T, effector-to-target ratio.
Figure 2.
Figure 2.
Augmented functional activity of CMVpp65CTLs responding to pentadecapeptides containing overlapping epitopes presented by HLA class I and class II alleles. Comparison of IFN-γ CD3+ T-cell responses to the CMVpp65 15-mer peptide pool in CMVpp65CTLs derived from donors who coinherited both the class I and class II HLA alleles presented epitopes in an overlapping 15-mer peptide vs responses in donors generating both CD4+ and CD8+ T cells to CMVpp65 peptides that do not overlap or to individuals generating only a CD8+ CMVpp65–specific T-cell response.
Figure 3.
Figure 3.
Comparison of dominant vs nondominant responses in 41 individuals who generated IFN-γ+ CMVpp65CTLs against >1 peptide epitope. Dominant responses are quantitated above the x-axis; nondominant responses from the same individual are quantitated below the axis. The HLA restriction of each T cell is above (dominant) or below (nondominant) each response compared.
Figure 4.
Figure 4.
Function of HLA A*0201 vs HLA B*3501 restricted CMVpp65CTLs. Comparison of NLV-specific HLA A*0201 restricted vs IPSI-specific HLA B*3501 restricted CMVpp65CTL lines in the bank, as to proportions of IFN-γ+ peptide-specific T cells in each line and the cytotoxic activity of each line against autologous phytohemagglutinin-stimulated blasts loaded with the targeted CMVpp65 peptide.
Figure 5.
Figure 5.
Comparisons of HLA-B*3501–restricted, IPSI peptide–specific vs HLA A*0201– restricted, NLV peptide–specific T cells as to their avidity for cognate peptide loaded on autologous DCs and their capacity to lyse autologous CMV-infected DCs. CMV-specific CTLs were incubated for 16 hours with autologous DCs loaded with10-fold serial dilutions of CMV-pp65 subpools containing the HLA A0201 peptide epitope NLVPMVATV or the HLA B3501 epitope IPSINVHHY. IFN-γ expression was then assessed by using an intracellular cytokine assay: (A) Representative flow cytometric analysis after stimulation of NLV or IPS-specific T cells with serial dilutions of cognate peptide. (B) Peptide dose–response curves for the experiment shown in panel A. (C) Chromium-51 release assay showing specific lysis by HLA-B35–restricted CTLs and HLA-A02–restricted CTLs of autologous DCs infected or not with the TB40E endotheliotropic clinical strain of HCMV. E:T, effector-to-target ratio.
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